Sample records for bacterial small-subunit rrna

  1. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  2. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences

    NASA Technical Reports Server (NTRS)

    Robison-Cox, J. F.; Bateson, M. M.; Ward, D. M.

    1995-01-01

    Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed.

  3. Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: evidence for a novel site of streptomycin resistance in the small subunit rRNA.

    PubMed

    Gauthier, A; Turmel, M; Lemieux, C

    1988-10-01

    A major obstacle to our understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.

  4. Regulation of Plasmodium yoelii oocyst development by strain- and stage-specific small-subunit rRNA.

    PubMed

    Qi, Yanwei; Zhu, Feng; Eastman, Richard T; Fu, Young; Zilversmit, Martine; Pattaradilokrat, Sittiporn; Hong, Lingxian; Liu, Shengfa; McCutchan, Thomas F; Pan, Weiqing; Xu, Wenyue; Li, Jian; Huang, Fusheng; Su, Xin-zhuan

    2015-03-10

    One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD--characterized as having small oocysts and lacking infective sporozoites--was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the

  5. Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene.

    PubMed Central

    Li, S; Cullen, D; Hjort, M; Spear, R; Andrews, J H

    1996-01-01

    Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities. PMID:8633850

  6. Ribosomal small subunit domains radiate from a central core

    NASA Astrophysics Data System (ADS)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  7. Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

    PubMed

    Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

    2013-09-01

    Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

  8. Theileria sp. Infections Associated with Bovine Fatalities in the United States Confirmed by Small-Subunit rRNA Gene Analyses of Blood and Tick Samples

    PubMed Central

    Chae, Joon-seok; Levy, Michael; Hunt, John; Schlater, Jack; Snider, Glen; Waghela, Suryakant D.; Holman, Patricia J.; Wagner, G. Gale

    1999-01-01

    Theileria sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and in Amblyomma americanum and Dermacentor variabilis ticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D Theileria SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovine Theileria isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis. PMID:10449501

  9. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    NASA Technical Reports Server (NTRS)

    Winker, S.; Woese, C. R.

    1991-01-01

    The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

  10. Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli.

    PubMed

    Smith, Brian A; Gupta, Neha; Denny, Kevin; Culver, Gloria M

    2018-06-08

    Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

    PubMed

    van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

    1992-10-01

    The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

  12. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  13. The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

    PubMed Central

    De Wachter, R; Neefs, J M; Goris, A; Van de Peer, Y

    1992-01-01

    The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing. PMID:1561081

  14. Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

    PubMed

    Viscogliosi, E; Edgcomb, V P; Gerbod, D; Noël, C; Delgado-Viscogliosi, P

    1999-12-01

    The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

  15. Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

    NASA Technical Reports Server (NTRS)

    Viscogliosi, E.; Edgcomb, V. P.; Gerbod, D.; Noel, C.; Delgado-Viscogliosi, P.; Sogin, M. L. (Principal Investigator)

    1999-01-01

    The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

  16. Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China

    PubMed Central

    Liu, Qing; Zhu, Shenghua; Mizuno, Sahoko; Kimura, Masatsugu; Liu, Peina; Isomura, Shin; Wang, Xingzhen; Kawamoto, Fumihiko

    1998-01-01

    By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia. PMID:9774600

  17. Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

    PubMed

    Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

    2010-12-01

    Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

  18. Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

    PubMed Central

    Peccia, Jordan; Marchand, Eric A.; Silverstein, Joann; Hernandez, Mark

    2000-01-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using 32P radiolabels, probe specificity was characterized by hybridization dissociation temperature (Td) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined Tds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris. PMID:10877807

  19. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical

  20. Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

    PubMed Central

    Moy, Terence I.; Silver, Pamela A.

    1999-01-01

    After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

  1. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    PubMed

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  2. Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

    PubMed Central

    Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

    1994-01-01

    Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

  3. Phylogeny of the Haplosporidia (Eukaryota: Alveolata) based on small subunit ribosomal RNA gene sequence.

    PubMed

    Flores, B S; Siddall, M E; Burreson, E M

    1996-08-01

    The phylogenetic position of the phylum Haplosporidia was investigated with the complete small subunit rRNA gene sequences from 5 species in the phylum: Haplosporidium nelsoni and Haplosporidium costale, parasites of the eastern oyster Crassostrea virginica; Haplosporidium louisiana, a parasite of the mudcrab Panopeus herbstii; Minchinia teredinis, a parasite of shipworms (Teredo spp.) and Urosporidium crescens, a hyperparasite found in metacercariae of the trematode Megalophallus sp. in the blue crab, Callinectes sapidus. Multiple alignments of small subunit rRNA gene sequences included the 5 haplosporidian taxa and 14 taxa in the alveolate phyla Ciliophora, Dinoflagellida, and Apicomplexa. Maximum parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the alveolate clade, as a taxon of equal rank with the other 3 alveolate phyla, and as a sister taxon to the clade composed of the phyla Dinoflagellida and Apicomplexa. Transversionally weighted parsimony placed the haplosporidians as a sister taxon to the ciliates. A separate analysis focused on the relationships of species in the genus Haplosporidium. Analyses were conducted with the haplosporidians as a functional ingroup, using each of the alveolate phyla individually as functional outgroups. The results indicated that species in the genus Haplosporidium do not form a monophyletic assemblage. As such, the present morphological criteria for distinguishing the genera Haplosporidium and Minchinia are insufficient.

  4. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  5. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

    PubMed

    Römling, Ute; Galperin, Michael Y

    2015-09-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    PubMed Central

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  7. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    PubMed Central

    Petrova, Olga E.; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

    2017-01-01

    Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested. PMID:28117413

  8. Sequence data for two large-subunit rRNA genes from an Asian strain of Alexandrium catenella.

    PubMed Central

    Yeung, P K; Kong, K F; Wong, F T; Wong, J T

    1996-01-01

    PCR generated two distinct products from a toxic isolate of Alexandrium catenella, which had been taken from Dai Ya Bay (southern China), by using primers for large-subunit rRNA. This pattern is distinct from published data for North American Alexandrium species. Sequences of the two products suggest that the smaller was generated by a deletion event. Single-cell PCR generated the same pattern, confirming that the two products were not the results from different individuals. PMID:8900010

  9. Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples

    PubMed Central

    Yan, Yong-Wei; Zou, Bin; Zhu, Ting; Hozzein, Wael N.

    2017-01-01

    RNA-seq-based SSU (small subunit) rRNA (ribosomal RNA) analysis has provided a better understanding of potentially active microbial community within environments. However, for RNA-seq library construction, high quantities of purified RNA are typically required. We propose a modified RNA-seq method for SSU rRNA-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10–100 ng) and does not require a DNA removal step. The method was initially tested on three mock communities synthesized with enriched SSU rRNA of archaeal, bacterial and fungal isolates at different ratios, and was subsequently used for environmental samples of high or low biomass. For high-biomass salt-marsh sediments, enriched SSU rRNA and total nucleic acid-derived RNA-seq datasets revealed highly consistent community compositions for all of the SSU rRNA sequences, and as much as 46.4%-59.5% of 16S rRNA sequences were suitable for OTU (operational taxonomic unit)-based community and diversity analyses with complete coverage of V1-V2 regions. OTU-based community structures for the two datasets were also highly consistent with those determined by all of the 16S rRNA reads. For low-biomass samples, total nucleic acid-derived RNA-seq datasets were analyzed, and highly active bacterial taxa were also identified by the OTU-based method, notably including members of the previously underestimated genus Nitrospira and phylum Acidobacteria in tap water, members of the phylum Actinobacteria on a shower curtain, and members of the phylum Cyanobacteria on leaf surfaces. More than half of the bacterial 16S rRNA sequences covered the complete region of primer 8F, and non-coverage rates as high as 38.7% were obtained for phylum-unclassified sequences, providing many opportunities to identify novel bacterial taxa. This modified RNA-seq method will provide a better snapshot of diverse

  10. Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli.

    PubMed

    Koay, Teng Wei; Wong, Hann Ling; Lim, Boon Hoe

    2016-11-26

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

  11. Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

    PubMed

    Mundus, D; Wollenzien, P

    1998-11-01

    Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

  12. Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA.

    PubMed

    Goggin, C L; Barker, S C

    1993-07-01

    Parasites of the genus Perkinsus destroy marine molluscs worldwide. Their phylogenetic position within the kingdom Protista is controversial. Nucleotide sequence data (1792 bp) from the small subunit rRNA gene of Perkinsus sp. from Anadara trapezia (Mollusca: Bivalvia) from Moreton Bay, Queensland, was used to examine the phylogenetic affinities of this enigmatic genus. These data were aligned with nucleotide sequences from 6 apicomplexans, 3 ciliates, 3 flagellates, a dinoflagellate, 3 fungi, maize and human. Phylogenetic trees were constructed after analysis with maximum parsimony and distance matrix methods. Our analyses indicate that Perkinsus is phylogenetically closer to dinoflagellates and to coccidean and piroplasm apicomplexans than to fungi or flagellates.

  13. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA.

    PubMed

    Medina, M; Collins, A G; Silberman, J D; Sogin, M L

    2001-08-14

    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino-Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira-Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of a monophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.

  14. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Medina, Monica; Collins, Allen G.; Silberman, Jeffrey

    2001-06-21

    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino-Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira-Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combinedmore » data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of amonophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.« less

  15. 16S rRNA beacons for bacterial monitoring during human space missions.

    PubMed

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  16. Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association.

    PubMed

    Pulk, Arto; Maiväli, Ulo; Remme, Jaanus

    2006-05-01

    The ribosome consists of two unequal subunits, which associate via numerous intersubunit contacts. Medium-resolution structural studies have led to grouping of the intersubunit contacts into 12 directly visualizable intersubunit bridges. Most of the intersubunit interactions involve RNA. We have used an RNA modification interference approach to determine Escherichia coli 16S rRNA positions that are essential for the association of functionally active 70S ribosomes. Modification of the N1 position of A702, A1418, and A1483 with DMS, and of the N3 position of U793, U1414, and U1495 with CMCT in 30S subunits strongly interferes with 70S ribosome formation. Five of these positions localize into previously recognized intersubunit bridges, namely, B2a (U1495), B2b (U793), B3 (A1483), B5 (A1418), and B7a (A702). The remaining position displaying interference, U1414, forms a base pair with G1486, which is a part of bridge B3. We contend that these five intersubunit bridges are essential for reassociation of the 70S ribosome, thus forming the functional core of the intersubunit contacts.

  17. Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association

    PubMed Central

    Pulk, Arto; Maiväli, Ülo; Remme, Jaanus

    2006-01-01

    The ribosome consists of two unequal subunits, which associate via numerous intersubunit contacts. Medium-resolution structural studies have led to grouping of the intersubunit contacts into 12 directly visualizable intersubunit bridges. Most of the intersubunit interactions involve RNA. We have used an RNA modification interference approach to determine Escherichia coli 16S rRNA positions that are essential for the association of functionally active 70S ribosomes. Modification of the N1 position of A702, A1418, and A1483 with DMS, and of the N3 position of U793, U1414, and U1495 with CMCT in 30S subunits strongly interferes with 70S ribosome formation. Five of these positions localize into previously recognized intersubunit bridges, namely, B2a (U1495), B2b (U793), B3 (A1483), B5 (A1418), and B7a (A702). The remaining position displaying interference, U1414, forms a base pair with G1486, which is a part of bridge B3. We contend that these five intersubunit bridges are essential for reassociation of the 70S ribosome, thus forming the functional core of the intersubunit contacts. PMID:16556933

  18. Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

    PubMed

    Gerbod, D; Edgcomb, V P; Noël, C; Delgado-Viscogliosi, P; Viscogliosi, E

    2000-09-01

    Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

  19. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    PubMed

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  20. Caryotricha minuta (Xu et al., 2008) nov. comb., a unique marine ciliate (Protista, Ciliophora, Spirotrichea), with phylogenetic analysis of the ambiguous genus Caryotricha inferred from the small-subunit rRNA gene sequence.

    PubMed

    Miao, Miao; Shao, Chen; Jiang, Jiamei; Li, Liqiong; Stoeck, Thorsten; Song, Weibo

    2009-02-01

    A population of Kiitricha minuta Xu et al., 2008, a small kiitrichid ciliate, was isolated from a brackish water sample in Jiaozhou Bay, Qingdao, northern China. After comparison of its morphology and infraciliature, it is believed that this morphotype should be assigned to the genus Caryotricha; hence, a new combination is suggested, Caryotricha minuta (Xu et al., 2008) nov. comb. The small-subunit (SSU) rRNA gene sequence was determined in order to elucidate the phylogenetic position of this poorly known, ambiguous genus. The organism can be clearly separated from its congener, Caryotricha convexa Kahl, 1932, by the extremely shortened ventral cirral rows in the posterior ends. Based on the data available, an improved diagnosis is given for the genus: marine Kiitrichidae with prominent buccal field; two highly developed undulating membranes; non-grouped, uniform cirral rows on both ventral and dorsal sides; enlarged transverse cirri present, which are the only differentiated cirri; marginal cirri not present; one short migratory row located posterior to buccal field; structure of dorsal kineties generally in Kiitricha pattern. The sequence of the SSU rRNA gene of C. minuta differs by 13 % from that of Kiitricha marina. Molecular phylogenetic analyses (Bayesian inference, least squares, neighbour joining, maximum parsimony) indicate that Caryotricha, together with Kiitricha, diverges at a deep level from all other spirotrichs. Its branching position is between Phacodiniidia and Licnophoridia. The results strongly support the distinct separation of the Kiitricha-Caryotricha clade, which always branches basal to the Stichotrichia-Hypotrichia-Oligotrichia-Choreotrichia assemblage. These results also confirm the previous hypothesis that the Kiitricha-Caryotricha group, long assumed to be a close relation to the euplotids, represents a taxon at subclass level within the spirotrichs.

  1. Small bowel bacterial overgrowth

    MedlinePlus

    Overgrowth - intestinal bacteria; Bacterial overgrowth - intestine; Small intestinal bacterial overgrowth; SIBO ... intestine does not have a high number of bacteria. Excess bacteria in the small intestine may use ...

  2. Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

    PubMed

    Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

    2002-12-01

    The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

  3. Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

    PubMed Central

    Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

    2002-01-01

    The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. PMID:12515387

  4. Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia.

    PubMed

    Hershey, H P; Schwartz, L J; Gale, J P; Abell, L M

    1999-07-01

    Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

  5. Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and “Nonbacterial” Prostatitis

    PubMed Central

    Tanner, Michael A.; Shoskes, Daniel; Shahed, Asha; Pace, Norman R.

    1999-01-01

    The etiology of chronic prostatitis syndromes in men is controversial, particularly when positive cultures for established uropathogens are lacking. Although identification of bacteria in prostatic fluid has relied on cultivation and microscopy, most microorganisms in the environment, including some human pathogens, are resistant to cultivation. We report here on an rRNA-based molecular phylogenetic approach to the identification of bacteria in prostate fluid from prostatitis patients. Positive bacterial signals were seen for 65% of patients with chronic prostatitis overall. Seven of 11 patients with bacterial signals but none of 6 patients without bacterial signals were cured with antibiotic-based therapy. Results indicate the occurrence in the prostate fluid of a wide spectrum of bacterial species representing several genera. Most rRNA genes were closely related to those of species belonging to the genera Corynebacterium, Staphylococcus, Peptostreptococcus, Streptococcus, and Escherichia. Unexpectedly, a wide diversity of Corynebacterium species was found in high proportion compared to the proportions of other bacterial species found. A subset of these 16S rRNA sequences represent those of undescribed species on the basis of their positions in phylogenetic trees. These uncharacterized organisms were not detected in control samples, suggesting that the organisms have a role in the disease or are the consequence of the disease. These studies show that microorganisms associated with prostatitis generally occur as complex microbial communities that differ between patients. The results also indicate that microbial communities distinct from those associated with prostatitis may occur at low levels in normal prostatic fluid. PMID:10325338

  6. Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

    PubMed Central

    Weiss, Andy; Shaw, Lindsey N.

    2015-01-01

    The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

  7. Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

    PubMed Central

    Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

    2012-01-01

    Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

  8. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  9. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

    PubMed

    Nath, B Surendra; Gupta, S K; Bajpai, A K

    2012-12-01

    The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

  10. Interactions of 2’-O-methyl oligoribonucleotides with the RNA models of the 30S subunit A-site

    PubMed Central

    Jasiński, Maciej; Kulik, Marta; Wojciechowska, Monika; Stolarski, Ryszard

    2018-01-01

    Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2’-O-methylated (2’-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2’-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2’-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2’-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2’-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred

  11. Vertical Distribution of Bacterial Communities in the Indian Ocean as Revealed by Analyses of 16S rRNA and nasA Genes.

    PubMed

    Jiang, Xuexia; Jiao, Nianzhi

    2016-09-01

    Bacteria play an important role in the marine biogeochemical cycles. However, research on the bacterial community structure of the Indian Ocean is scarce, particularly within the vertical dimension. In this study, we investigated the bacterial diversity of the pelagic, mesopelagic and bathypelagic zones of the southwestern Indian Ocean (50.46°E, 37.71°S). The clone libraries constructed by 16S rRNA gene sequence revealed that most phylotypes retrieved from the Indian Ocean were highly divergent from those retrieved from other oceans. Vertical differences were observed based on the analysis of natural bacterial community populations derived from the 16S rRNA gene sequences. Based on the analysis of the nasA gene sequences from GenBank database, a pair of general primers was developed and used to amplify the bacterial nitrate-assimilating populations. Environmental factors play an important role in mediating the bacterial communities in the Indian Ocean revealed by canonical correlation analysis.

  12. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1987-05-01

    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

  13. Genotypic variation of Pneumocystis jirovecii isolates in India based on sequence diversity at mitochondrial large subunit rRNA.

    PubMed

    Gupta, Rashmi; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Samantaray, Jyotish Chandra; Kumar, Lalit; Kabra, Sushil Kumar; Luthra, Kalpana; Sreenivas, Vishnubhatla; Iyer, Venkateswaran K

    2011-03-01

    Pneumocystis pneumonia (PCP), a common and serious opportunistic infection in immunocompromised patients, is caused by Pneumocystis jirovecii (formerly known as Pneumocystis carinii f. sp. hominis). The aim of the present study was to describe the prevalence and distribution of genotypes of P. jirovecii based on sequence polymorphisms at mitochondrial large subunit ribosomal RNA (mt LSU rRNA) region in both HIV and non-HIV immunocompromised individuals with a positive PCR result for PCP in a tertiary health care centre in northern India. From January 2005 to October 2008, 50 patients [22 HIV-seropositive individuals, 10 post-renal transplant (PRT) recipients, 3 cancer patients, and 15 patients with various other kinds of immunosuppression] were found to be positive for P. jirovecii using PCR at the mt LSU rRNA gene. Genotyping of the positive samples was performed at the mt LSU rRNA locus. Genotype 2 was the most common accounting for 42% of total types. This was followed by the genotypes 3 (24%), 1 (20%), and 4 (8%). Mixed infection was observed in 3 cases (6%). The rates of genotype distribution were similar in HIV-seropositive individuals, cancer patients, and in patients with other kinds of immunosuppression. In the PRT recipients, genotype 1 was the most prevalent type (80%). This is the first study describing the prevalence of genotypes in HIV-infected and HIV-uninfected, immunocompromised patients based on the mt LSU rRNA gene from the Indian subcontinent. The most prevalent genotype observed was type 2 in contrast to many studies from other parts of the world where genotype 1 was the most prevalent type, suggesting geographical variation. Copyright © 2010 Elsevier GmbH. All rights reserved.

  14. Detection of a mixed infection in a culture-negative brain abscess by broad-spectrum bacterial 16S rRNA gene PCR.

    PubMed

    Keller, Peter M; Rampini, Silvana K; Bloemberg, Guido V

    2010-06-01

    We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.

  15. Differential targeting of Gbetagamma-subunit signaling with small molecules.

    PubMed

    Bonacci, Tabetha M; Mathews, Jennifer L; Yuan, Chujun; Lehmann, David M; Malik, Sundeep; Wu, Dianqing; Font, Jose L; Bidlack, Jean M; Smrcka, Alan V

    2006-04-21

    G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  16. Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA.

    PubMed

    Hughes, J M

    1996-06-21

    The U3 nucleolar RNA has a remarkably wide phyletic distribution extending from the Eukarya to the Archaea. It functions in maturation of the small subunit (SSU) rRNA through a mechanism which is as yet unknown but which involves base-pairing with pre-rRNA. The most conserved part of U3 is within 30 nucleotides of the 5' end, but as yet no function for this domain has been proposed. Elements within this domain are complementary to highly conserved sequences in the SSU rRNA which, in the mature form, fold into a universally conserved pseudoknot. The nature of the complementarity suggests a novel mechanism for U3 function whereby U3 facilitates correct folding of the pseudoknot. Wide phylogenetic comparison provides compelling evidence in support of the interaction in that significant complementary changes have taken place, particularly in the archaeon Sulfolobus, which maintain the base-pairing. Base-substitution mutations in yeast U3 designed to disrupt the base-pairing indicate that the interaction is probably essential. These include cold-sensitivity mutations which exhibit phenotypes similar to U3-depletion, but without impairment of the AO processing step, which occurs within the 5' ETS. These phenotypes are consistent with the destabilization of SSU precursors and partial impairment of the processing steps A1, at the 5' ETS/18 S boundary, and A2, within the ITS1.

  17. Description of Eurystomatella sinica n. gen., n. sp., with establishment of a new family Eurystomatellidae n. fam. (Protista, Ciliophora, Scuticociliatia) and analyses of its phylogeny inferred from sequences of the small-subunit rRNA gene.

    PubMed

    Miao, Miao; Wang, Yangang; Song, Weibo; Clamp, John C; Al-Rasheid, Khaled A S

    2010-02-01

    Recently, an undescribed marine ciliate was isolated from China. Investigation of its morphology and infraciliature revealed it as an undescribed species representing a new genus, Eurystomatella n. gen., the type of the new family Eurystomatellidae n. fam. The new family is defined by close-set, apically positioned oral membranelles and a dominant buccal field that is surrounded by an almost completely circular paroral membrane. The new genus is defined by having a small oral membranelle 1 (M1), bipartite M2 and well-developed M3, a body surface faintly sculptured with a silverline system in a quadrangular, reticulate pattern and a cytostome located at the anterior third of a large buccal field. The type species of the new genus, Eurystomatella sinica n. sp., is a morphologically unique form that is defined mainly by the combination of a conspicuously flattened body, several caudal cilia, extremely long cilia associated with the buccal apparatus and a contractile vacuole located subcaudally. According to phylogenetic analyses of small-subunit (SSU) rRNA gene sequences, Eurystomatella clusters with the genus Cyclidium, as a sister group to the family Pleuronematidae. The great divergence in both buccal and somatic ciliature between Eurystomatella and all other known scuticociliates supports the establishment of a new family for Eurystomatella.

  18. Terminator oligo blocking efficiently eliminates rRNA from Drosophila small RNA sequencing libraries.

    PubMed

    Wickersheim, Michelle L; Blumenstiel, Justin P

    2013-11-01

    A large number of methods are available to deplete ribosomal RNA reads from high-throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nucleotide 2S rRNA. Here we demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to 5' adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction in a simple and inexpensive way. This depletion is highly specific and is achieved with minimal perturbation of miRNA and piRNA profiles.

  19. From learning taxonomies to phylogenetic learning: integration of 16S rRNA gene data into FAME-based bacterial classification.

    PubMed

    Slabbinck, Bram; Waegeman, Willem; Dawyndt, Peter; De Vos, Paul; De Baets, Bernard

    2010-01-30

    Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering algorithms on FAME data or on 16S rRNA gene data. The knowledge gained from the tree can then be used to evaluate FAME-based classifiers, resulting in a novel framework for bacterial species classification. In view of learning in a taxonomic framework, we consider two types of trees. First, a FAME tree is constructed with a supervised divisive clustering algorithm. Subsequently, based on 16S rRNA gene sequence analysis, phylogenetic trees are inferred by the NJ and UPGMA methods. In this second approach, the species classification problem is based on the combination of two different types of data. Herein, 16S rRNA gene sequence data is used for phylogenetic tree inference and the corresponding binary tree splits are learned based on FAME data. We call this learning approach 'phylogenetic learning'. Supervised Random Forest models are developed to train the classification tasks in a stratified cross-validation setting. In this way, better classification results are obtained for species that are typically hard to distinguish by a single or flat multi-class classification model. FAME-based bacterial species classification is successfully evaluated in a taxonomic framework. Although the proposed approach does not improve the overall accuracy compared to flat multi-class classification, it has some distinct advantages. First, it has better capabilities for distinguishing species on which flat multi-class classification fails. Secondly, the hierarchical classification structure allows to easily evaluate and visualize the resolution of FAME data for the discrimination of bacterial

  20. From learning taxonomies to phylogenetic learning: Integration of 16S rRNA gene data into FAME-based bacterial classification

    PubMed Central

    2010-01-01

    Background Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering algorithms on FAME data or on 16S rRNA gene data. The knowledge gained from the tree can then be used to evaluate FAME-based classifiers, resulting in a novel framework for bacterial species classification. Results In view of learning in a taxonomic framework, we consider two types of trees. First, a FAME tree is constructed with a supervised divisive clustering algorithm. Subsequently, based on 16S rRNA gene sequence analysis, phylogenetic trees are inferred by the NJ and UPGMA methods. In this second approach, the species classification problem is based on the combination of two different types of data. Herein, 16S rRNA gene sequence data is used for phylogenetic tree inference and the corresponding binary tree splits are learned based on FAME data. We call this learning approach 'phylogenetic learning'. Supervised Random Forest models are developed to train the classification tasks in a stratified cross-validation setting. In this way, better classification results are obtained for species that are typically hard to distinguish by a single or flat multi-class classification model. Conclusions FAME-based bacterial species classification is successfully evaluated in a taxonomic framework. Although the proposed approach does not improve the overall accuracy compared to flat multi-class classification, it has some distinct advantages. First, it has better capabilities for distinguishing species on which flat multi-class classification fails. Secondly, the hierarchical classification structure allows to easily evaluate and visualize the resolution of FAME data for

  1. Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

    PubMed Central

    Yang, F; Curran, S C; Li, L S; Avarbock, D; Graf, J D; Chua, M M; Lu, G; Salem, J; Rubin, H

    1997-01-01

    Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon. PMID:9335290

  2. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

    PubMed

    Suchodolski, Jan S; Dowd, Scot E; Wilke, Vicky; Steiner, Jörg M; Jergens, Albert E

    2012-01-01

    Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

  3. Detection of a Mixed Infection in a Culture-Negative Brain Abscess by Broad-Spectrum Bacterial 16S rRNA Gene PCR ▿ †

    PubMed Central

    Keller, Peter M.; Rampini, Silvana K.; Bloemberg, Guido V.

    2010-01-01

    We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis. PMID:20392909

  4. Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

    PubMed Central

    Dalmay, Tamas

    2018-01-01

    RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues. PMID:29474379

  5. Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys

    PubMed Central

    Werner, Jeffrey J; Koren, Omry; Hugenholtz, Philip; DeSantis, Todd Z; Walters, William A; Caporaso, J Gregory; Angenent, Largus T; Knight, Rob; Ley, Ruth E

    2012-01-01

    Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases. PMID:21716311

  6. Profiling bacterial diversity in a limestone cave of the western Loess Plateau of China

    PubMed Central

    Wu, Yucheng; Tan, Liangcheng; Liu, Wuxing; Wang, Baozhan; Wang, Jianjun; Cai, Yanjun; Lin, Xiangui

    2015-01-01

    Bacteria and archaea sustain subsurface cave ecosystems by dominating primary production and fueling biogeochemical cyclings, despite the permanent darkness and shortage of nutrients. However, the heterogeneity and underlying mechanism of microbial diversity in caves, in particular those well connect to surface environment are largely unexplored. In this study, we examined the bacterial abundance and composition in Jinjia Cave, a small and shallow limestone cave located on the western Loess Plateau of China, by enumerating and pyrosequencing small subunit rRNA genes. The results clearly reveal the contrasting bacterial community compositions in relation to cave habitat types, i.e., rock wall deposit, aquatic sediment, and sinkhole soil, which are differentially connected to the surface environment. The deposits on the cave walls were dominated by putative cave-specific bacterial lineages within the γ-Proteobacteria or Actinobacteria that are routinely found on cave rocks around the world. In addition, sequence identity with known functional groups suggests enrichments of chemolithotrophic bacteria potentially involved in autotrophic C fixation and inorganic N transformation on rock surfaces. By contrast, bacterial communities in aquatic sediments were more closely related to those in the overlying soils. This is consistent with the similarity in elemental composition between the cave sediment and the overlying soil, implicating the influence of mineral chemistry on cave microhabitat and bacterial composition. These findings provide compelling molecular evidence of the bacterial community heterogeneity in an East Asian cave, which might be controlled by both subsurface and surface environments. PMID:25870592

  7. Profiling bacterial diversity in a limestone cave of the western Loess Plateau of China.

    PubMed

    Wu, Yucheng; Tan, Liangcheng; Liu, Wuxing; Wang, Baozhan; Wang, Jianjun; Cai, Yanjun; Lin, Xiangui

    2015-01-01

    Bacteria and archaea sustain subsurface cave ecosystems by dominating primary production and fueling biogeochemical cyclings, despite the permanent darkness and shortage of nutrients. However, the heterogeneity and underlying mechanism of microbial diversity in caves, in particular those well connect to surface environment are largely unexplored. In this study, we examined the bacterial abundance and composition in Jinjia Cave, a small and shallow limestone cave located on the western Loess Plateau of China, by enumerating and pyrosequencing small subunit rRNA genes. The results clearly reveal the contrasting bacterial community compositions in relation to cave habitat types, i.e., rock wall deposit, aquatic sediment, and sinkhole soil, which are differentially connected to the surface environment. The deposits on the cave walls were dominated by putative cave-specific bacterial lineages within the γ-Proteobacteria or Actinobacteria that are routinely found on cave rocks around the world. In addition, sequence identity with known functional groups suggests enrichments of chemolithotrophic bacteria potentially involved in autotrophic C fixation and inorganic N transformation on rock surfaces. By contrast, bacterial communities in aquatic sediments were more closely related to those in the overlying soils. This is consistent with the similarity in elemental composition between the cave sediment and the overlying soil, implicating the influence of mineral chemistry on cave microhabitat and bacterial composition. These findings provide compelling molecular evidence of the bacterial community heterogeneity in an East Asian cave, which might be controlled by both subsurface and surface environments.

  8. A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

    PubMed

    Helbling, Damian E; Johnson, David R; Lee, Tae Kwon; Scheidegger, Andreas; Fenner, Kathrin

    2015-03-01

    The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific

  9. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  10. Accurate, Rapid Taxonomic Classification of Fungal Large-Subunit rRNA Genes

    PubMed Central

    Liu, Kuan-Liang; Porras-Alfaro, Andrea; Eichorst, Stephanie A.

    2012-01-01

    Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp). PMID:22194300

  11. Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

    PubMed Central

    Smith, Maria W.; Meskauskas, Arturas; Wang, Pinger; Sergiev, Petr V.; Dinman, Jonathan D.

    2001-01-01

    rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome. PMID:11713264

  12. Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

    PubMed Central

    Chiaruttini, C; Expert-Bezançon, A; Hayes, D; Ehresmann, B

    1982-01-01

    1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide. Images PMID:6760129

  13. 16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

    PubMed Central

    Suchodolski, Jan S.; Dowd, Scot E.; Wilke, Vicky; Steiner, Jörg M.; Jergens, Albert E.

    2012-01-01

    Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation. PMID:22720094

  14. Phylogenetic relationship of psychoactive fungi based on rRNA gene for a large subunit and their identification using the TaqMan assay (II).

    PubMed

    Maruyama, Takuro; Kawahara, Nobuo; Yokoyama, Kazumasa; Makino, Yukiko; Fukiharu, Toshimitsu; Goda, Yukihiro

    2006-11-10

    "Magic mushroom (MM)" is the name most commonly given to psychoactive fungi containing the hallucinogenic components: psilocin (1) and psilocybin (2). We investigated the rRNA gene (internal transcribed spacer (ITS) and large subunit (LSU)) of two Panaeolus species and four Psilocybe species fungi (of these, two are non-psilocybin species). On the basis of sequence alignment, we improved the identification system developed in our previous study. In this paper, we describe the new system capable of distinguishing MMs from non-psilocybin Psilocybe species, its application data and the phylogeny of MM species.

  15. Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

    PubMed

    Połka, Justyna; Rebecchi, Annalisa; Pisacane, Vincenza; Morelli, Lorenzo; Puglisi, Edoardo

    2015-04-01

    The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential

  16. Biogeographic Patterns Between Bacterial Phyllosphere Communities of the Southern Magnolia (Magnolia grandiflora) in a Small Forest.

    PubMed

    Stone, Bram W G; Jackson, Colin R

    2016-05-01

    The phyllosphere presents a unique system of discrete and easily replicable surfaces colonized primarily by bacteria. However, the biogeography of bacteria in the phyllosphere is little understood, especially at small to intermediate scales. Bacterial communities on the leaves of 91 southern magnolia (Magnolia grandiflora) trees 1-452 m apart in a small forest plot were analyzed and fragments of the 16S ribosomal RNA (rRNA) gene sequenced using the Illumina platform. Assemblages were dominated by members of the Alphaproteobacteria, Bacteroidetes, and Acidobacteria. Patterns in community composition were measured by both relative abundance (theta) and presence-absence (Jaccard) dissimilarity metrics. Distance-based Moran's eigenvector map analyses of the distance-decay relationship found a significant, positive relationship between each dissimilarity metric and significant eigenfunctions derived from geographic distance between trees, indicating trees that were closer together had more similar bacterial phyllosphere communities. Indirect gradient analyses revealed that several environmental parameters (canopy cover, tree elevation, and the slope and aspect of the ground beneath trees) were significantly related to multivariate ordination scores based on relative bacterial sequence abundances; however, these relationships were not significant when looking at the incidence of bacterial taxa. This suggests that bacterial growth and abundance in the phyllosphere is shaped by different assembly mechanisms than bacterial presence or absence. More broadly, this study demonstrates that the distance-decay relationship applies to phyllosphere communities at local scales, and that environmental parameters as well as neutral forces may both influence spatial patterns in the phyllosphere.

  17. Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania.

    PubMed

    Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

    2012-12-01

    Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.

  18. Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

    PubMed Central

    Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

    2012-01-01

    Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved. PMID:22767185

  19. Small subunit ribosomal RNA genes of tabanids and hippoboscids (Diptera: Brachycera): evolutionary relationships and comparison with other Diptera.

    PubMed

    Carreno, R A; Barta, J R

    1998-11-01

    The small subunit ribosomal RNA (SSU rRNA) genes of hippoboscid (Ornithoica vicina Walker) and tabanid (Chrysops niger Macquart) Diptera were sequenced to determine their phylogenetic position within the order and to determine whether or not extensive hypervariable regions in this gene are widespread in the Diptera. A parsimony analysis of an alignment containing 8 dipteran sequences produced a single most parsimonious tree that placed O. vicina as sister group to Drosophila melanogaster Meigen. The tabanid Chrysops niger was sister group to the asilomorphan taxa, and the sister group to the Brachycera was a Tipula sp. although this relationship was not supported by bootstrap analysis. The hippoboscid and tabanid sequences contain extensive hypervariable regions in the V2, V4, V6, and V7 regions as do other Diptera. When these regions of the alignment were excluded from the phylogenetic analysis, a single most parsimonious tree was found. This tree had an identical overall topology to the tree obtained from the total data set. The hypervariable regions in parts of the dipteran SSU rRNA genes were more extensive in the nematocerous dipteran sequences used in this study than in the other dipteran representatives; these hypervariable regions may be of more utility in inferring relationship among species and subspecies than at the suprageneric level.

  20. Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

    PubMed Central

    Ahmed, Tofayel; Shi, Jian

    2017-01-01

    Abstract Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c. PMID:28582576

  1. Primer selection impacts specific population abundances but not community dynamics in a monthly time-series 16S rRNA gene amplicon analysis of coastal marine bacterioplankton.

    PubMed

    Wear, Emma K; Wilbanks, Elizabeth G; Nelson, Craig E; Carlson, Craig A

    2018-03-09

    Primers targeting the 16S small subunit ribosomal RNA marker gene, used to characterize bacterial and archaeal communities, have recently been re-evaluated for marine planktonic habitats. To investigate whether primer selection affects the ecological interpretation of bacterioplankton populations and community dynamics, amplicon sequencing with four primer sets targeting several hypervariable regions of the 16S rRNA gene was conducted on both mock communities constructed from cloned 16S rRNA genes and a time-series of DNA samples from the temperate coastal Santa Barbara Channel. Ecological interpretations of community structure (delineation of depth and seasonality, correlations with environmental factors) were similar across primer sets, while population dynamics varied. We observed substantial differences in relative abundances of taxa known to be poorly resolved by some primer sets, such as Thaumarchaeota and SAR11, and unexpected taxa including Roseobacter clades. Though the magnitude of relative abundances of common OTUs differed between primer sets, the relative abundances of the OTUs were nonetheless strongly correlated. We do not endorse one primer set but rather enumerate strengths and weaknesses to facilitate selection appropriate to a system or experimental goal. While 16S rRNA gene primer bias suggests caution in assessing quantitative population dynamics, community dynamics appear robust across studies using different primers. © 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Sequence and Secondary Structure of the Mitochondrial Small-Subunit rRNA V4, V6, and V9 Domains Reveal Highly Species-Specific Variations within the Genus Agrocybe

    PubMed Central

    Gonzalez, Patrice; Labarère, Jacques

    1998-01-01

    A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The

  3. Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe.

    PubMed

    Gonzalez, P; Labarère, J

    1998-11-01

    A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The

  4. Directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S13 using tethered Fe(II).

    PubMed Central

    Heilek, G M; Noller, H F

    1996-01-01

    Directed hydroxyl radical probing was used to probe the rRNA neighborhood around protein S13 in the 30S ribosomal subunit. The unique cysteine at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) was then assembled into 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleavage of the RNA backbone in two localized regions of the 3' major domain of 16S rRNA. One region spans nt 1308-1333 and is close to a site previously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 by previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results place both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit. PMID:8718688

  5. Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors.

    PubMed

    Walcher, Petra; Mayr, Ulrike B; Azimpour-Tabrizi, Chakameh; Eko, Francis O; Jechlinger, Wolfgang; Mayrhofer, Peter; Alefantis, Tim; Mujer, Cesar V; DelVecchio, Vito G; Lubitz, Werner

    2004-12-01

    The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties. The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells. The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously. Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA. Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells. There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response. All can be used for subunit antigen to design new vaccine candidates with particle presentation technology. In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens. Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy. The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.

  6. Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

    PubMed Central

    Liiv, Aivar; Karitkina, Diana; Maiväli, Ülo; Remme, Jaanus

    2005-01-01

    Background The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. Results We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. Conclusion Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes. PMID:16053518

  7. Molecular analysis of 16S rRNA genes identifies potentially periodontal pathogenic bacteria and archaea in the plaque of partially erupted third molars.

    PubMed

    Mansfield, J M; Campbell, J H; Bhandari, A R; Jesionowski, A M; Vickerman, M M

    2012-07-01

    Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites. Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present. Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes and Spirochaetes. Dialister invisus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis, Prevotella denticola, Tannerella forsythia, and Treponema denticola, which have been associated with periodontal disease, were found in significantly greater abundance in pericoronal compared with incisor sites. Dialister invisus and F nucleatum were found in greater abundance in sites exhibiting clinical symptoms. The archaeal species, Methanobrevibacter oralis, which has been associated with severe periodontitis, was found in 3 symptomatic patients. These findings have provided new insights into the complex microbiota of pericoronitis. Several bacterial and archaeal species implicated in periodontal disease were recovered in greater incidence and abundance from the plaque of partially erupted third molars compared with incisors, supporting the hypothesis that the pericoronal region may provide a favored niche for periodontal pathogens in otherwise healthy mouths. Copyright © 2012 American Association of Oral and

  8. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    USDA-ARS?s Scientific Manuscript database

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  9. Structural insights into cell cycle control by essential GTPase Era.

    PubMed

    Ji, Xinhua

    Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides ( 1530 GAUCACCUCC 1539 ) near the 3' end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Era's GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.

  10. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  11. The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose.

    PubMed

    Treede, Irina; Jakobsen, Lene; Kirpekar, Finn; Vester, Birte; Weitnauer, Gabriele; Bechthold, Andreas; Douthwaite, Stephen

    2003-07-01

    Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tü57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N-methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2'-O-ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N-1 of the G2535 base and the 2'-hydroxyl of U2479 are separated by approximately 10 A, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.

  12. The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP.

    PubMed

    Sardana, Richa; White, Joshua P; Johnson, Arlen W

    2013-06-01

    Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

  13. A Comparison of Structural and Evolutionary Attributes of Escherichia coli and Thermus thermophilus Small Ribosomal Subunits: Signatures of Thermal Adaptation

    PubMed Central

    Mallik, Saurav; Kundu, Sudip

    2013-01-01

    Here we compare the structural and evolutionary attributes of Thermus thermophilus and Escherichia coli small ribosomal subunits (SSU). Our results indicate that with few exceptions, thermophilic 16S ribosomal RNA (16S rRNA) is densely packed compared to that of mesophilic at most of the analogous spatial regions. In addition, we have located species-specific cavity clusters (SSCCs) in both species. E. coli SSCCs are numerous and larger compared to T. thermophilus SSCCs, which again indicates densely packed thermophilic 16S rRNA. Thermophilic ribosomal proteins (r-proteins) have longer disordered regions than their mesophilic homologs and they experience larger disorder-to-order transitions during SSU-assembly. This is reflected in the predicted higher conformational changes of thermophilic r-proteins compared to their mesophilic homologs during SSU-assembly. This high conformational change of thermophilic r-proteins may help them to associate with the 16S ribosomal RNA with high complementary interfaces, larger interface areas, and denser molecular contacts, compared to those of mesophilic. Thus, thermophilic protein-rRNA interfaces are tightly associated with 16S rRNA than their mesophilic homologs. Densely packed 16S rRNA interior and tight protein-rRNA binding of T. thermophilus (compared to those of E. coli) are likely the signatures of its thermal adaptation. We have found a linear correlation between the free energy of protein-RNA interface formation, interface size, and square of conformational changes, which is followed in both prokaryotic and eukaryotic SSU. Disorder is associated with high protein-RNA interface polarity. We have found an evolutionary tendency to maintain high polarity (thereby disorder) at protein-rRNA interfaces, than that at rest of the protein structures. However, some proteins exhibit exceptions to this general trend. PMID:23940533

  14. On the Mg(2+) binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2015-10-01

    F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg(2+) concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg(2+) binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg(2+) ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg(2+) dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg(2+) binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg(2+) binding motif by site directed mutagenesis, which also can be used to crystallize Mg(2+) dependent ε subunits in future. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. rRNA fragmentation induced by a yeast killer toxin.

    PubMed

    Kast, Alene; Klassen, Roland; Meinhardt, Friedhelm

    2014-02-01

    Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins. © 2013 John Wiley & Sons Ltd.

  16. Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

    PubMed

    Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

    2011-10-01

    The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

  17. Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

    PubMed Central

    Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

    2001-01-01

    The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

  18. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

    PubMed Central

    Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

    2016-01-01

    Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

  19. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGES

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; ...

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  20. Identification of Cellulose-Responsive Bacterial and Fungal Communities in Geographically and Edaphically Different Soils by Using Stable Isotope Probing

    PubMed Central

    Eichorst, Stephanie A.

    2012-01-01

    Many bacteria and fungi are known to degrade cellulose in culture, but their combined response to cellulose in different soils is unknown. Replicate soil microcosms amended with [13C]cellulose were used to identify bacterial and fungal communities responsive to cellulose in five geographically and edaphically different soils. The diversity and composition of the cellulose-responsive communities were assessed by DNA-stable isotope probing combined with Sanger sequencing of small-subunit and large-subunit rRNA genes for the bacterial and fungal communities, respectively. In each soil, the 13C-enriched, cellulose-responsive communities were of distinct composition compared to the original soil community or 12C-nonenriched communities. The composition of cellulose-responsive taxa, as identified by sequence operational taxonomic unit (OTU) similarity, differed in each soil. When OTUs were grouped at the bacterial order level, we found that members of the Burkholderiales, Caulobacteriales, Rhizobiales, Sphingobacteriales, Xanthomonadales, and the subdivision 1 Acidobacteria were prevalent in the 13C-enriched DNA in at least three of the soils. The cellulose-responsive fungi were identified as members of the Trichocladium, Chaetomium, Dactylaria, and Arthrobotrys genera, along with two novel Ascomycota clusters, unique to one soil. Although similarities were identified in higher-level taxa among some soils, the composition of cellulose-responsive bacteria and fungi was generally unique to a certain soil type, suggesting a strong potential influence of multiple edaphic factors in shaping the community. PMID:22287013

  1. Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

    PubMed

    Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

    2009-07-07

    Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

  2. Composition and Metabolic Activities of the Bacterial Community in Shrimp Sauce at the Flavor-Forming Stage of Fermentation As Revealed by Metatranscriptome and 16S rRNA Gene Sequencings.

    PubMed

    Duan, Shan; Hu, Xiaoxi; Li, Mengru; Miao, Jianyin; Du, Jinghe; Wu, Rongli

    2016-03-30

    The bacterial community and the metabolic activities involved at the flavor-forming stage during the fermentation of shrimp sauce were investigated using metatranscriptome and 16S rRNA gene sequencings. Results showed that the abundance of Tetragenococcus was 95.1%. Tetragenococcus halophilus was identified in 520 of 588 transcripts annotated in the Nr database. Activation of the citrate cycle and oxidative phosphorylation, along with the absence of lactate dehydrogenase gene expression, in T. halophilus suggests that T. halophilus probably underwent aerobic metabolism during shrimp sauce fermentation. The metabolism of amino acids, production of peptidase, and degradation of limonene and pinene were very active in T. halophilus. Carnobacterium, Pseudomonas, Escherichia, Staphylococcus, Bacillus, and Clostridium were also metabolically active, although present in very small populations. Enterococcus, Abiotrophia, Streptococcus, and Lactobacillus were detected in metatranscriptome sequencing, but not in 16S rRNA gene sequencing. Many minor taxa showed no gene expression, suggesting that they were in dormant status.

  3. Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.

    PubMed

    Gladden, John M; Allgaier, Martin; Miller, Christopher S; Hazen, Terry C; VanderGheynst, Jean S; Hugenholtz, Philip; Simmons, Blake A; Singer, Steven W

    2011-08-15

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  4. Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

    PubMed

    Guo, Liliang; Sui, Zhenghong; Zhang, Shu; Ren, Yuanyuan; Liu, Yuan

    2015-04-01

    Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode. © 2015 IUMS.

  5. How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?

    PubMed

    Rodriguez-R, Luis M; Castro, Juan C; Kyrpides, Nikos C; Cole, James R; Tiedje, James M; Konstantinidis, Konstantinos T

    2018-03-15

    The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas , Burkholderia , Escherichia , Campylobacter , and Citrobacter These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary. IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA

  6. Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide.

    PubMed

    Yang, Yang; Gao, Tian; Xu, Mengjun; Dong, Jie; Li, Hanxiao; Wang, Pengfei; Li, Gezi; Guo, Tiancai; Kang, Guozhang; Wang, Yonghua

    2017-03-01

    ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat ( Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.

  7. hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

    PubMed Central

    Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

    2015-01-01

    Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

  8. mRNA bound to the 30S subunit is a HigB toxin substrate

    PubMed Central

    Schureck, Marc A.; Maehigashi, Tatsuya; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

    2016-01-01

    Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs. PMID:27307497

  9. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

    PubMed

    Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

    2009-08-01

    Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

  10. Mineral Type and Solution Chemistry Affect the Structure and Composition of Actively Growing Bacterial Communities as Revealed by Bromodeoxyuridine Immunocapture and 16S rRNA Pyrosequencing.

    PubMed

    Kelly, L C; Colin, Y; Turpault, M-P; Uroz, S

    2016-08-01

    Understanding how minerals affect bacterial communities and their in situ activities in relation to environmental conditions are central issues in soil microbial ecology, as minerals represent essential reservoirs of inorganic nutrients for the biosphere. To determine the impact of mineral type and solution chemistry on soil bacterial communities, we compared the diversity, composition, and functional abilities of a soil bacterial community incubated in presence/absence of different mineral types (apatite, biotite, obsidian). Microcosms were prepared containing different liquid culture media devoid of particular essential nutrients, the nutrients provided only in the introduced minerals and therefore only available to the microbial community through mineral dissolution by biotic and/or abiotic processes. By combining functional screening of bacterial isolates and community analysis by bromodeoxyuridine DNA immunocapture and 16S rRNA gene pyrosequencing, we demonstrated that bacterial communities were mainly impacted by the solution chemistry at the taxonomic level and by the mineral type at the functional level. Metabolically active bacterial communities varied with solution chemistry and mineral type. Burkholderia were significantly enriched in the obsidian treatment compared to the biotite treatment and were the most effective isolates at solubilizing phosphorous or mobilizing iron, in all the treatments. A detailed analysis revealed that the 16S rRNA gene sequences of the OTUs or isolated strains assigned as Burkholderia in our study showed high homology with effective mineral-weathering bacteria previously recovered from the same experimental site.

  11. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

    PubMed

    Matsutani, Sachiko

    2004-08-09

    In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

  12. Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

    PubMed

    Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

    2011-02-01

    Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

  13. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

    PubMed Central

    Soltanieh, Sahar; Osheim, Yvonne N.; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L.

    2014-01-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. PMID:25535329

  15. Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes.

    PubMed

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-09-24

    Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

  16. Identification of the bacterial etiology of culture-negative endocarditis by amplification and sequencing of a small ribosomal RNA gene.

    PubMed

    Khulordava, Irakli; Miller, Geraldine; Haas, David; Li, Haijing; McKinsey, Joel; Vanderende, Daniel; Tang, Yi-Wei

    2003-05-01

    We report two cases of culture-negative bacterial endocarditis in which the organisms were identified by amplification and sequencing of the bacterial 16S rRNA gene. These results support an important role for polymerase chain reaction followed by direct sequencing to determine the etiology of culture-negative bacterial endocarditis and to guide appropriate antimicrobial therapy.

  17. Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

    PubMed

    Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

    2015-07-01

    The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

    PubMed Central

    Kiparisov, S.; Sergiev, P. V.; Dontsova, O. A.; Petrov, A.; Meskauskas, A.; Dinman, J. D.

    2005-01-01

    5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression. PMID:16047201

  19. Bacterial communities in the small intestine respond differently to those in the caecum and colon in mice fed low- and high-fat diets

    PubMed Central

    Campbell, Sara; Moreau, Michael; Patel, Falshruti; Brooks, Andrew I.; Zhou, Yin Xiu; Häggblom, Max M.; Storch, Judith

    2017-01-01

    Bacterial communities in the mouse caecum and faeces are known to be altered by changes in dietary fat. The microbiota of the mouse small intestine, by contrast, has not been extensively profiled and it is unclear whether small intestinal bacterial communities shift with dietary fat levels. We compared the microbiota in the small intestine, caecum and colon in mice fed a low-fat (LF) or high-fat (HF) diet using 16S rRNA gene sequencing. The relative abundance of major phyla in the small intestine, Bacteriodetes, Firmicutes and Proteobacteria, was similar to that in the caecum and colon; the relative abundance of Verrucomicrobia was significantly reduced in the small intestine compared to the large intestine. Several genera were uniquely detected in the small intestine and included the aerotolerant anaerobe, Lactobacillus spp. The most abundant genera in the small intestine were accounted for by anaerobic bacteria and were identical to those identified in the large intestine. An HF diet was associated with significant weight gain and adiposity and with changes in the bacterial communities throughout the intestine, with changes in the small intestine differing from those in the caecum and colon. Prominent Gram-negative bacteria including genera of the phylum Bacteroidetes and a genus of Proteobacteria significantly changed in the large intestine. The mechanistic links between these changes and the development of obesity, perhaps involving metabolic endotoxemia, remain to be determined. PMID:28742010

  20. COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

    PubMed

    Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

    2011-09-01

    The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

  1. Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

    PubMed

    Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

    2008-04-01

    In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

  2. Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis.

    PubMed

    Li, Meiju; Zhou, Mi; Adamowicz, Elizabeth; Basarab, John A; Guan, Le Luo

    2012-02-24

    Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (∼1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing.

    PubMed

    Zuo, Yu; Xie, Wenfang; Pang, Yue; Li, Tiesong; Li, Qingwei; Li, Yingying

    2017-01-01

    The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.

  4. Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting.

    PubMed

    Valiente Moro, Claire; Thioulouse, Jean; Chauve, Claude; Normand, Philippe; Zenner, Lionel

    2009-01-01

    Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, "Candidatus Cardinium hertigii" and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control.

  5. Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

    PubMed Central

    Ehresmann, C; Moine, H; Mougel, M; Dondon, J; Grunberg-Manago, M; Ebel, J P; Ehresmann, B

    1986-01-01

    The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far. Images PMID:2425339

  6. Rifaximin Reduces Markers of Inflammation and Bacterial 16S rRNA in Zambian Adults with Hepatosplenic Schistosomiasis: A Randomized Control Trial.

    PubMed

    Sinkala, Edford; Zyambo, Kanekwa; Besa, Ellen; Kaonga, Patrick; Nsokolo, Bright; Kayamba, Violet; Vinikoor, Michael; Zulu, Rabison; Bwalya, Martin; Foster, Graham R; Kelly, Paul

    2018-04-01

    Cirrhosis is the dominant cause of portal hypertension globally but may be overshadowed by hepatosplenic schistosomiasis (HSS) in the tropics. In Zambia, schistosomiasis seroprevalence can reach 88% in endemic areas. Bacterial translocation (BT) drives portal hypertension in cirrhosis contributing to mortality but remains unexplored in HSS. Rifaximin, a non-absorbable antibiotic may reduce BT. We aimed to explore the influence of rifaximin on BT, inflammation, and fibrosis in HSS. In this phase II open-label trial (ISRCTN67590499), 186 patients with HSS in Zambia were evaluated and 85 were randomized to standard care with or without rifaximin for 42 days. Changes in markers of inflammation, BT, and fibrosis were the primary outcomes. BT was measured using plasma 16S rRNA, lipopolysaccharide-binding protein, and lipopolysaccharide, whereas hyaluronan was used to measure fibrosis. Tumor necrosis factor receptor 1 (TNFR1) and soluble cluster of differentiation 14 (sCD14) assessed inflammation. 16S rRNA reduced from baseline (median 146 copies/µL, interquartile range [IQR] 9, 537) to day 42 in the rifaximin group (median 63 copies/µL, IQR 12, 196), P < 0.01. The rise in sCD14 was lower ( P < 0.01) in the rifaximin group (median rise 122 ng/mL, IQR-184, 783) than in the non-rifaximin group (median rise 832 ng/mL, IQR 530, 967). TNFR1 decreased ( P < 0.01) in the rifaximin group (median -39 ng/mL IQR-306, 563) but increased in the non-rifaximin group (median 166 ng/mL, IQR 3, 337). Other markers remained unaffected. Rifaximin led to a reduction of inflammatory markers and bacterial 16S rRNA which may implicate BT in the inflammation in HSS.

  7. Rifaximin Reduces Markers of Inflammation and Bacterial 16S rRNA in Zambian Adults with Hepatosplenic Schistosomiasis: A Randomized Control Trial

    PubMed Central

    Sinkala, Edford; Zyambo, Kanekwa; Besa, Ellen; Kaonga, Patrick; Nsokolo, Bright; Kayamba, Violet; Vinikoor, Michael; Zulu, Rabison; Bwalya, Martin; Foster, Graham R.; Kelly, Paul

    2018-01-01

    Abstract. Cirrhosis is the dominant cause of portal hypertension globally but may be overshadowed by hepatosplenic schistosomiasis (HSS) in the tropics. In Zambia, schistosomiasis seroprevalence can reach 88% in endemic areas. Bacterial translocation (BT) drives portal hypertension in cirrhosis contributing to mortality but remains unexplored in HSS. Rifaximin, a non-absorbable antibiotic may reduce BT. We aimed to explore the influence of rifaximin on BT, inflammation, and fibrosis in HSS. In this phase II open-label trial (ISRCTN67590499), 186 patients with HSS in Zambia were evaluated and 85 were randomized to standard care with or without rifaximin for 42 days. Changes in markers of inflammation, BT, and fibrosis were the primary outcomes. BT was measured using plasma 16S rRNA, lipopolysaccharide-binding protein, and lipopolysaccharide, whereas hyaluronan was used to measure fibrosis. Tumor necrosis factor receptor 1 (TNFR1) and soluble cluster of differentiation 14 (sCD14) assessed inflammation. 16S rRNA reduced from baseline (median 146 copies/µL, interquartile range [IQR] 9, 537) to day 42 in the rifaximin group (median 63 copies/µL, IQR 12, 196), P < 0.01. The rise in sCD14 was lower (P < 0.01) in the rifaximin group (median rise 122 ng/mL, IQR-184, 783) than in the non-rifaximin group (median rise 832 ng/mL, IQR 530, 967). TNFR1 decreased (P < 0.01) in the rifaximin group (median -39 ng/mL IQR-306, 563) but increased in the non-rifaximin group (median 166 ng/mL, IQR 3, 337). Other markers remained unaffected. Rifaximin led to a reduction of inflammatory markers and bacterial 16S rRNA which may implicate BT in the inflammation in HSS. PMID:29436337

  8. Bioprospecting from cultivable bacterial communities of marine sediment and invertebrates from the underexplored Ubatuba region of Brazil.

    PubMed

    Tangerina, Marcelo M P; Correa, Hebelin; Haltli, Brad; Vilegas, Wagner; Kerr, Russell G

    2017-01-01

    Shrimp fisheries along the Brazilian coast have significant environmental impact due to high by-catch rates (21 kg per kilogram of shrimp). Typically discarded, by-catch contains many invertebrates that may host a great variety of bacterial genera, some of which may produce bioactive natural products with biotechnological applications. Therefore, to utilize by-catch that is usually discarded we explored the biotechnological potential of culturable bacteria of two abundant by-catch invertebrate species, the snail Olivancillaria urceus and the sea star Luidia senegalensis. Sediment from the collection area was also investigated. Utilizing multiple isolation approaches, 134 isolates were obtained from the invertebrates and sediment. Small-subunit rRNA (16S) gene sequencing revealed that the isolates belonged to Proteobacteria, Firmicutes and Actinobacteria phyla and were distributed among 28 genera. Several genera known for their capacity to produce bioactive natural products (Micromonospora, Streptomyces, Serinicoccus and Verrucosispora) were retrieved from the invertebrate samples. To query the bacterial isolates for their ability to produce bioactive metabolites, all strains were fermented and fermentation extracts profiled by UP LC-HRMS and tested for antimicrobial activity. Four strains exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus warneri.

  9. Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

    PubMed Central

    Dourado, Manuella Nóbrega; Andreote, Fernando Dini; Dini-Andreote, Francisco; Conti, Raphael; Araújo, Janete Magali; Araújo, Welington Luiz

    2012-01-01

    The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant. PMID:22481887

  10. Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association.

    PubMed

    Dourado, Manuella Nóbrega; Andreote, Fernando Dini; Dini-Andreote, Francisco; Conti, Raphael; Araújo, Janete Magali; Araújo, Welington Luiz

    2012-01-01

    The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

  11. Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: comparison of two different approaches in clinical practice.

    PubMed

    Jenkins, Claire; Ling, Clare L; Ciesielczuk, Holly L; Lockwood, Julianne; Hopkins, Susan; McHugh, Timothy D; Gillespie, Stephen H; Kibbler, Christopher C

    2012-04-01

    Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culture-negative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

  12. Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing.

    PubMed

    Bolhuis, Henk; Stal, Lucas J

    2011-11-01

    Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.

  13. Modular structure of the full-length DNA gyrase B subunit revealed by small-angle X-ray scattering.

    PubMed

    Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

    2007-03-01

    DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research.

  14. Bacterial 16S rRNA gene analysis revealed that bacteria related to Arcobacter spp. constitute an abundant and common component of the oyster microbiota (Tiostrea chilensis).

    PubMed

    Romero, J; García-Varela, M; Laclette, J P; Espejo, R T

    2002-11-01

    To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.

  15. Bacterial DNA detected on pathologically changed heart valves using 16S rRNA gene amplification.

    PubMed

    Chalupova, Miroslava; Skalova, Anna; Hajek, Tomas; Geigerova, Lenka; Kralova, Dana; Liska, Pavel; Hecova, Hana; Molacek, Jiri; Hrabak, Jaroslav

    2018-05-22

    Nowadays, dental diseases are one of the most common illnesses in the world. Some of them can lead to translocation of oral bacteria to the bloodstream causing intermittent bacteraemia. Therefore, a potential association between oral infection and cardiovascular diseases has been discussed in recent years as a result of adhesion of oral microbes to the heart valves. The aim of this study was to detect oral bacteria on pathologically changed heart valves not caused by infective endocarditis. In the study, patients with pathologically changed heart valves were involved. Samples of heart valves removed during heart valve replacement surgery were cut into two parts. One aliquot was cultivated aerobically and anaerobically. Bacterial DNA was extracted using Ultra-Deep Microbiome Prep (Molzym GmbH, Bremen, Germany) followed by a 16S rRNA gene PCR amplification using Mastermix 16S Complete kit (Molzym GmbH, Bremen, Germany). Positive PCR products were sequenced and the sequences were analyzed using BLAST database ( http://www.ncbi.nlm.nih/BLAST ). During the study period, 41 samples were processed. Bacterial DNA of the following bacteria was detected in 21 samples: Cutibacterium acnes (formerly Propionibacterium acnes) (n = 11; 52.38% of patients with positive bacterial DNA detection), Staphylococcus sp. (n = 9; 42.86%), Streptococcus sp. (n = 1; 4.76%), Streptococcus sanguinis (n = 4; 19.05%), Streptococcus oralis (n = 1; 4.76%), Carnobacterium sp. (n = 1; 4.76%), Bacillus sp. (n = 2; 9.52%), and Bergeyella sp. (n = 1; 4.76%). In nine samples, multiple bacteria were found. Our results showed significant appearance of bacteria on pathologically changed heart valves in patients with no symptoms of infective endocarditis.

  16. Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Jun; McCann, Kathleen L.; Qiu, Chen

    Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C’-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease,more » Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.« less

  17. Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia).

    PubMed

    Douzery, E; Catzeflis, F M

    1995-11-01

    The complete 12S rRNA gene has been sequenced in 4 Ungulata (hoofed eutherians) and 1 marsupial and compared to 38 available mammalian sequences in order to investigate the molecular evolution of the mitochondrial small-subunit ribosomal RNA molecule. Ungulata were represented by one artiodactyl (the collared peccary, Tayassu tajacu, suborder Suiformes), two perissodactyls (the Grevy's zebra, Equus grevyi, suborder Hippomorpha; the white rhinoceros, Ceratotherium simum, suborder Ceratomorpha), and one hyracoid (the tree hyrax, Dendrohyrax dorsalis). The fifth species was a marsupial, the eastern gray kangaroo (Macropus giganteus). Several transition/transversion biases characterized the pattern of changes between mammalian 12S rRNA molecules. A bias toward transitions was found among 12S rRNA sequences of Ungulata, illustrating the general bias exhibited by ribosomal and protein-encoding genes of the mitochondrial genome. The derivation of a mammalian 12S rRNA secondary structure model from the comparison of 43 eutherian and marsupial sequences evidenced a pronounced bias against transversions in stems. Moreover, transversional compensatory changes were rare events within double-stranded regions of the ribosomal RNA. Evolutionary characteristics of the 12S rRNA were compared with those of the nuclear 18S and 28S rRNAs. From a phylogenetic point of view, transitions, transversions and indels in stems as well as transversional and indels events in loops gave congruent results for comparisons within orders. Some compensatory changes in double-stranded regions and some indels in single-stranded regions also constituted diagnostic events. The 12S rRNA molecule confirmed the monophyly of infraorder Pecora and order Cetacea and demonstrated the monophyly of the suborder Ruminantia was not supported and the branching pattern between Cetacea and the artiodacytyl suborders Ruminantia and Suiformes was not established. The monophyly of the order Perissodactyla was evidenced

  18. Novel division level bacterial diversity in a Yellowstone hot spring.

    PubMed

    Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

    1998-01-01

    A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These

  19. Morphology and small-subunit rRNA gene sequences of two novel marine ciliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov. (Protista, Ciliophora, Scuticociliatia), with an improved diagnosis of the genus Uronemella.

    PubMed

    Pan, Xuming; Zhu, Mingzhuang; Ma, Honggang; Al-Rasheid, Khaled A S; Hu, Xiaozhong

    2013-09-01

    The morphology and infraciliature of two novel marine scuticociliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov., collected from sandy beaches at Qingdao, China, were investigated using live observation and protargol-staining methods. Metanophrys orientalis spec. nov. is distinguished by the following characteristics: marine habitat and a slender to elongate oval body with pointed anterior end and rounded caudal end, in vivo about 25-50 µm long; buccal field about a quarter to a third of body length; nine or ten somatic kineties with dikinetids approximately in anterior half of body, monokinetids in posterior half; membranelles 1 and 2 almost equal in length and composed of two and three longitudinal rows of kinetids respectively; paroral membrane with zigzag structure extending anteriorly to middle portion of membranelle 2; contractile vacuole pore located at posterior end of somatic kinety 1. The genus Uronemella is redefined as follows: marine form with an elongate-elliptical or inverted pear-shaped body; apical plate conspicuous; buccal field about two-thirds of body length, cytostome subequatorially located; oral apparatus Uronema-like; somatic kineties comprising a mixture of dikinetids and monokinetids. Uronemella sinensis spec. nov. is recognized by having an elongate-elliptical body with truncated apical frontal plate, size in vivo about 25-35 × 15-20 µm, nine or ten somatic kineties, membranelle 1 consisting of two or three basal bodies, contractile vacuole pore at posterior end of somatic kinety 1. This study also compared the small-subunit rRNA gene sequences of these two species with other closely related species to show the sequence divergence, which ranged from 3.53 to 9.60%. Phylogenetic analyses support the contention that the genus Uronemella is monophyletic, while Metanophrys is non-monophyletic.

  20. Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys

    PubMed Central

    Berry, David; Gutierrez, Tony

    2017-01-01

    Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys. PMID:28567035

  1. Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.

    PubMed

    Berry, David; Gutierrez, Tony

    2017-01-01

    Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira , and Thalassolituus , as well as the Alphaproteobacterial genus Thalassospira . Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.

  2. Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

    PubMed Central

    Duthoit, Frédérique; Godon, Jean-Jacques; Montel, Marie-Christine

    2003-01-01

    Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products. PMID:12839752

  3. The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing

    PubMed Central

    2009-01-01

    Background Recent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing. Results Pyrosequencing revealed a previously unrecognized species richness in the canine small intestine. Ten bacterial phyla were identified. Microbial populations were phylogenetically more similar during tylosin treatment. However, a remarkable inter-individual response was observed for specific taxa. Fusobacteria, Bacteroidales, and Moraxella tended to decrease. The proportions of Enterococcus-like organisms, Pasteurella spp., and Dietzia spp. increased significantly during tylosin administration (p < 0.05). The proportion of Escherichia coli-like organisms increased by day 28 (p = 0.04). These changes were not accompanied by any obvious clinical effects. On day 28, the phylogenetic composition of the microbiota was similar to day 0 in only 2 of 5 dogs. Bacterial diversity resembled the pre-treatment state in 3 of 5 dogs. Several bacterial taxa such as Spirochaetes, Streptomycetaceae, and Prevotellaceae failed to recover at day 28 (p < 0.05). Several bacterial groups considered to be sensitive to tylosin increased in their

  4. Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes.

    PubMed

    Milbury, Coren A; Lee, Jung C; Cannone, Jamie J; Gaffney, Patrick M; Gutell, Robin R

    2010-09-02

    Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

  5. Resistance and resilience responses of a range of soil eukaryote and bacterial taxa to fungicide application

    PubMed Central

    Howell, Christopher C.; Hilton, Sally; Semple, Kirk T.; Bending, Gary D.

    2014-01-01

    The application of plant protection products has the potential to significantly affect soil microbial community structure and function. However, the extent to which soil microbial communities from different trophic levels exhibit resistance and resilience to such compounds remains poorly understood. The resistance and resilience responses of a range of microbial communities (bacteria, fungi, archaea, pseudomonads, and nematodes) to different concentrations of the strobilurin fungicide, azoxystrobin were studied. A significant concentration-dependent decrease, and subsequent recovery in soil dehydrogenase activity was recorded, but no significant impact on total microbial biomass was observed. Impacts on specific microbial communities were studied using small subunit (SSU) rRNA terminal restriction fragment length polymorphism (T-RFLP) profiling using soil DNA and RNA. The application of azoxystrobin significantly affected fungal and nematode community structure and diversity but had no impact on other communities. Community impacts were more pronounced in the RNA-derived T-RFLP profiles than in the DNA-derived profiles. qPCR confirmed that azoxystrobin application significantly reduced fungal, but not bacterial, SSU rRNA gene copy number. Azoxystrobin application reduced the prevalence of ascomycete fungi, but increased the relative abundance of zygomycetes. Azoxystrobin amendment also reduced the relative abundance of nematodes in the order Enoplia, but stimulated a large increase in the relative abundance of nematodes from the order Araeolaimida. PMID:25048906

  6. Resistance and resilience responses of a range of soil eukaryote and bacterial taxa to fungicide application.

    PubMed

    Howell, Christopher C; Hilton, Sally; Semple, Kirk T; Bending, Gary D

    2014-10-01

    The application of plant protection products has the potential to significantly affect soil microbial community structure and function. However, the extent to which soil microbial communities from different trophic levels exhibit resistance and resilience to such compounds remains poorly understood. The resistance and resilience responses of a range of microbial communities (bacteria, fungi, archaea, pseudomonads, and nematodes) to different concentrations of the strobilurin fungicide, azoxystrobin were studied. A significant concentration-dependent decrease, and subsequent recovery in soil dehydrogenase activity was recorded, but no significant impact on total microbial biomass was observed. Impacts on specific microbial communities were studied using small subunit (SSU) rRNA terminal restriction fragment length polymorphism (T-RFLP) profiling using soil DNA and RNA. The application of azoxystrobin significantly affected fungal and nematode community structure and diversity but had no impact on other communities. Community impacts were more pronounced in the RNA-derived T-RFLP profiles than in the DNA-derived profiles. qPCR confirmed that azoxystrobin application significantly reduced fungal, but not bacterial, SSU rRNA gene copy number. Azoxystrobin application reduced the prevalence of ascomycete fungi, but increased the relative abundance of zygomycetes. Azoxystrobin amendment also reduced the relative abundance of nematodes in the order Enoplia, but stimulated a large increase in the relative abundance of nematodes from the order Araeolaimida. Copyright © 2014. Published by Elsevier Ltd.

  7. An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

    PubMed

    Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

    2009-10-19

    Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

  8. The gut bacterial communities associated with lab-raised and field-collected ants of Camponotus fragilis (Formicidae: Formicinae).

    PubMed

    He, Hong; Wei, Cong; Wheeler, Diana E

    2014-09-01

    Camponotus is the second largest ant genus and known to harbor the primary endosymbiotic bacteria of the genus Blochmannia. However, little is known about the effect of diet and environment changes on the gut bacterial communities of these ants. We investigated the intestinal bacterial communities in the lab-raised and field-collected ants of Camponotus fragilis which is found in the southwestern United States and northern reaches of Mexico. We determined the difference of gut bacterial composition and distribution among the crop, midgut, and hindgut of the two types of colonies. Number of bacterial species varied with the methods of detection and the source of the ants. Lab-raised ants yielded 12 and 11 species using classical microbial culture methods and small-subunit rRNA genes (16S rRNAs) polymerase chain reaction-restriction fragment-length polymorphism analysis, respectively. Field-collected ants yielded just 4 and 1-3 species using the same methods. Most gut bacterial species from the lab-raised ants were unevenly distributed among the crop, midgut, and hindgut, and each section had its own dominant bacterial species. Acetobacter was the prominent bacteria group in crop, accounting for about 55 % of the crop clone library. Blochmannia was the dominant species in midgut, nearly reaching 90 % of the midgut clone library. Pseudomonas aeruginosa dominated the hindgut, accounting for over 98 % of the hindgut clone library. P. aeruginosa was the only species common to all three sections. A comparison between lab-raised and field-collected ants, and comparison with other species, shows that gut bacterial communities vary with local environment and diet. The bacterial species identified here were most likely commensals with little effect on their hosts or mild pathogens deleterious to colony health.

  9. Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

    PubMed Central

    Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

    2015-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

  10. Structure of bacterial lipopolysaccharides.

    PubMed

    Caroff, Martine; Karibian, Doris

    2003-11-14

    Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.

  11. From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition.

    PubMed

    Porras-Alfaro, Andrea; Liu, Kuan-Liang; Kuske, Cheryl R; Xie, Gary

    2014-02-01

    We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5' section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.

  12. From Genus to Phylum: Large-Subunit and Internal Transcribed Spacer rRNA Operon Regions Show Similar Classification Accuracies Influenced by Database Composition

    PubMed Central

    Liu, Kuan-Liang; Kuske, Cheryl R.

    2014-01-01

    We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5′ section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets. PMID:24242255

  13. Structure of Rv1848 (UreA), the Mycobacterium tuberculosis urease γ subunit

    PubMed Central

    Habel, Jeff E.; Bursey, Evan H.; Rho, Beom-Seop; Kim, Chang-Yub; Segelke, Brent W.; Rupp, Bernhard; Park, Min S.; Terwilliger, Thomas C.; Hung, Li-Wei

    2010-01-01

    The crystal structure of the urease γ subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 Å resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (αβγ)3 composition observed for other bacterial ureases. The γ subunit may be of primary importance for the formation of the urease quaternary structure. PMID:20606272

  14. Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin.

    PubMed

    Witek, Marta A; Conn, Graeme L

    2014-09-01

    The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m(1)A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m(1)A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m(1)A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical enzyme is a functional 16S rRNA (m(1)A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Small-Scale Vertical Distribution of Bacterial Biomass and Diversity in Biological Soil Crusts from Arid Lands in the Colorado Plateau

    USGS Publications Warehouse

    Garcia-Pichel, F.; Johnson, S.L.; Youngkin, D.; Belnap, J.

    2003-01-01

    We characterized, at millimeter resolution, bacterial biomass, diversity, and vertical stratification of biological soil crusts in arid lands from the Colorado Plateau. Microscopic counts, extractable DNA, and plate counts of viable aerobic copiotrophs (VAC) revealed that the top centimeter of crusted soils contained atypically large bacterial populations, tenfold larger than those in uncrusted, deeper soils. The plate counts were not always consistent with more direct estimates of microbial biomass. Bacterial populations peaked at the immediate subsurface (1-2 mm) in light-appearing, young crusts, and at the surface (0-1 mm) in well-developed, dark crusts, which corresponds to the location of cyanobacterial populations. Bacterial abundance decreased with depth below these horizons. Spatially resolved DGGE fingerprints of Bacterial 16S rRNA genes demonstrated the presence of highly diverse natural communities, but we could detect neither trends with depth in bacterial richness or diversity, nor a difference in diversity indices between crust types. Fingerprints, however, revealed the presence of marked stratification in the structure of the microbial communities, probably a result of vertical gradients in physicochemical parameters. Sequencing and phylogenetic analyses indicated that most of the naturally occurring bacteria are novel types, with low sequence similarity (83-93%) to those available in public databases. DGGE analyses of the VAC populations indicated communities of lower diversity, with most types having sequences more than 94% similar to those in public databases. Our study indicates that soil crusts represent small-scale mantles of fertility in arid ecosystems, harboring vertically structured, little-known bacterial populations that are not well represented by standard cultivation methods.

  16. Molecular mechanics of 30S subunit head rotation.

    PubMed

    Mohan, Srividya; Donohue, John Paul; Noller, Harry F

    2014-09-16

    During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

  17. Molecular mechanics of 30S subunit head rotation

    PubMed Central

    Mohan, Srividya; Donohue, John Paul; Noller, Harry F.

    2014-01-01

    During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2. PMID:25187561

  18. Seasonal variation in detection of bacterial DNA in arthritic stifle joints of dogs with cranial cruciate ligament rupture using PCR amplification of the 16S rRNA gene.

    PubMed

    Muir, Peter; Fox, Robin; Wu, Qiang; Baker, Theresa A; Zitzer, Nina C; Hudson, Alan P; Manley, Paul A; Schaefer, Susan L; Hao, Zhengling

    2010-02-24

    An underappreciated cause and effect relationship between environmental bacteria and arthritis may exist. Previously, we found that stifle arthritis in dogs was associated with the presence of environmental bacteria within synovium. Cranial cruciate ligament rupture (CCLR) is often associated with stifle arthritis in dogs. We now wished to determine whether seasonal variation in detection of bacterial material may exist in affected dogs, and to also conduct analyses of both synovium and synovial fluid. We also wished to analyze a larger clone library of the 16S rRNA gene to further understanding of the microbial population in the canine stifle. Synovial biopsies were obtained from 117 affected dogs from January to December 2006. Using PCR, synovium and synovial fluid were tested for Borrelia burgdorferi and Stenotrophomonas maltophilia DNA. Broad-ranging 16S rRNA primers were also used and PCR products were cloned and sequenced for bacterial identification. Overall, 41% of arthritic canine stifle joints contained bacterial DNA. Detection of bacterial DNA in synovial fluid samples was increased, when compared with synovium (p<0.01). Detection rates were highest in the winter and spring and lowest in the summer period, suggesting environmental factors influence the risk of translocation to the stifle. Organisms detected were predominately Gram's negative Proteobacteria, particularly the orders Rhizobiales (32.8% of clones) and Burkholderiales (20.0% of clones), usually as part of a polymicrobial population. PCR-positivity was inversely correlated with severity of arthritis assessed radiographically and with dog age. Bacterial translocation to the canine stifle may be associated with changes to the indoor environment. Copyright 2009 Elsevier B.V. All rights reserved.

  19. Characterization of bacterial community associated with phytoplankton bloom in a eutrophic lake in South Norway using 16S rRNA gene amplicon sequence analysis.

    PubMed

    Parulekar, Niranjan Nitin; Kolekar, Pandurang; Jenkins, Andrew; Kleiven, Synne; Utkilen, Hans; Johansen, Anette; Sawant, Sangeeta; Kulkarni-Kale, Urmila; Kale, Mohan; Sæbø, Mona

    2017-01-01

    Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities.

  20. Role of small subunit in mediating assembly of red-type form I Rubisco.

    PubMed

    Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2015-01-09

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Bacterial taxa abundance pattern in an industrial wastewater treatment system determined by the full rRNA cycle approach.

    PubMed

    Figuerola, Eva L M; Erijman, Leonardo

    2007-07-01

    The description of the diversity and structure of microbial communities through quantification of the constituent populations is one of the major objectives in environmental microbiology. The implications of models for community assembly are practical as well as theoretical, because the extent of biodiversity is thought to influence the function of ecosystems. Current attempts to predict species diversity in different environments derive the numbers of individuals for each operational taxonomic unit (OTU) from the frequency of clones in 16S rDNA gene libraries, which are subjected to a number of inherent biases and artefacts. We show that diversity of the bacterial community present in a complex microbial ensemble can be estimated by fitting the data of the full-cycle rRNA approach to a model of species abundance distribution. Sequences from a 16S rDNA gene library from activated sludge were reliably assigned to OTUs at a genetic distance of 0.04. A group of 17 newly designed rRNA-targeted oligonucleotide probes were used to quantify by fluorescence in situ hybridization, OTUs represented with more than three clones in the 16S rDNA clone library. Cell abundance distribution was best described by a geometric series, after the goodness of fit was evaluated by the Kolmogorov-Smirnov test. Although a complete mechanistic understanding of all the ecological processes involved is still not feasible, describing the distribution pattern of a complex bacterial assemblage model can shed light on the way bacterial communities operate.

  2. Characterization of hydrocortisone biometabolites and 18S rRNA gene in Chlamydomonas reinhardtii cultures.

    PubMed

    Ghasemi, Younes; Rasoul-Amini, Sara; Morowvat, Mohammad Hossein; Raee, Mohammad Javad; Ghoshoon, Mohammad Bagher; Nouri, Fatemeh; Negintaji, Narges; Parvizi, Rezvan; Mosavi-Azam, Seyed Bagher

    2008-10-31

    A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 degrees C for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17 beta-Dihydroxyandrost-4-en-3-one (2), 11 beta-hydroxyandrost-4-en-3,17-dione (3), 11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one (4) and prednisolone (5) were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

  3. Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

    PubMed

    Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

    2018-01-01

    Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

  4. Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

    PubMed

    Haruta, Shin; Ueno, Shintaro; Egawa, Isao; Hashiguchi, Kazunori; Fujii, Akira; Nagano, Masanobu; Ishii, Masaharu; Igarashi, Yasuo

    2006-05-25

    Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

  5. Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine

    PubMed Central

    Guymon, Rebecca; Pomerantz, Steven C.; Ison, J. Nicholas; Crain, Pamela F.; McCloskey, James A.

    2007-01-01

    Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80°C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of ∼14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (M r 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii. PMID:17255199

  6. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

  7. Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing

    PubMed Central

    Sprockett, Daniel D.; Ammons, Christine G.; Tuttle, Marie S.

    2016-01-01

    Clinical diagnosis of infection in chronic wounds is currently limited to subjective clinical signs and culture-based methods that underestimate the complexity of wound microbial bioburden as revealed by DNA-based microbial identification methods. Here, we use 16S rRNA next generation sequencing and quantitative polymerase chain reaction to characterize weekly changes in bacterial load, community structure, and diversity associated with a chronic venous leg ulcer over the 15-week course of treatment and healing. Our DNA-based methods and detailed sampling scheme reveal that the bacterial bioburden of the wound is unexpectedly dynamic, including changes in the bacterial load and community structure that correlate with wound expansion, antibiotic therapy, and healing. We demonstrate that these multidimensional changes in bacterial bioburden can be summarized using swabs taken prior to debridement, and therefore, can be more easily collected serially than debridement or biopsy samples. Overall, this case illustrates the importance of detailed clinical indicators and longitudinal sampling to determine the pathogenic significance of chronic wound microbial dynamics and guide best use of antimicrobials for improvement of healing outcomes. PMID:25902876

  8. Q-PCR based bioburden assessment of drinking water throughout treatment and delivery to the International Space Station

    NASA Technical Reports Server (NTRS)

    Newcombe, David; Stuecker, Tara; La Duc, Myron; Venkateswaran, Kasthuri

    2005-01-01

    Previous studies indicated evidence of opportunistic pathogens samples obtained during missions to the International Space Station (ISS). This study utilized TaqMan quantitative PCR to determine specific gene abundance in potable and non-potable ISS waters. Probe and primer sets specific to the small subunit rRNA genes were used to elucidate overall bacterial rRNA gene numbers. while those specific for Burkholderia cepacia and Stenotrophomonas maltophilia were optimized and used to probe for the presence of these two opportunistic pathogens. This research builds upon previous microbial diversity studies of ISS water and demonstrates the utility of Q-PCR tool to examine water quality.

  9. Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

    PubMed

    Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

    2001-05-01

    Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

  10. Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

    PubMed Central

    Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

    2001-01-01

    Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding. PMID:11350033

  11. Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

    PubMed Central

    Simmon, Keith; Karaca, Dilek; Langeland, Nina; Wiker, Harald G.

    2012-01-01

    Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens. PMID:22278843

  12. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletionmore » and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.« less

  13. High-Throughput rRNA Gene Sequencing Reveals High
and Complex Bacterial Diversity Associated with
Brazilian Coffee Bean Fermentation

    PubMed Central

    Vinícius de Melo, Gilberto

    2018-01-01

    Summary Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

  14. Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454-pyrosequencing.

    PubMed

    Lopes, Ana R; Manaia, Célia M; Nunes, Olga C

    2014-03-01

    Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  15. Small intestinal bacterial overgrowth syndrome

    PubMed Central

    Bures, Jan; Cyrany, Jiri; Kohoutova, Darina; Förstl, Miroslav; Rejchrt, Stanislav; Kvetina, Jaroslav; Vorisek, Viktor; Kopacova, Marcela

    2010-01-01

    Human intestinal microbiota create a complex polymicrobial ecology. This is characterised by its high population density, wide diversity and complexity of interaction. Any dysbalance of this complex intestinal microbiome, both qualitative and quantitative, might have serious health consequence for a macro-organism, including small intestinal bacterial overgrowth syndrome (SIBO). SIBO is defined as an increase in the number and/or alteration in the type of bacteria in the upper gastrointestinal tract. There are several endogenous defence mechanisms for preventing bacterial overgrowth: gastric acid secretion, intestinal motility, intact ileo-caecal valve, immunoglobulins within intestinal secretion and bacteriostatic properties of pancreatic and biliary secretion. Aetiology of SIBO is usually complex, associated with disorders of protective antibacterial mechanisms (e.g. achlorhydria, pancreatic exocrine insufficiency, immunodeficiency syndromes), anatomical abnormalities (e.g. small intestinal obstruction, diverticula, fistulae, surgical blind loop, previous ileo-caecal resections) and/or motility disorders (e.g. scleroderma, autonomic neuropathy in diabetes mellitus, post-radiation enteropathy, small intestinal pseudo-obstruction). In some patients more than one factor may be involved. Symptoms related to SIBO are bloating, diarrhoea, malabsorption, weight loss and malnutrition. The gold standard for diagnosing SIBO is still microbial investigation of jejunal aspirates. Non-invasive hydrogen and methane breath tests are most commonly used for the diagnosis of SIBO using glucose or lactulose. Therapy for SIBO must be complex, addressing all causes, symptoms and complications, and fully individualised. It should include treatment of the underlying disease, nutritional support and cyclical gastro-intestinal selective antibiotics. Prognosis is usually serious, determined mostly by the underlying disease that led to SIBO. PMID:20572300

  16. Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

    PubMed Central

    Wasserman, Michael R.; Pulk, Arto; Zhou, Zhou; Altman, Roger B.; Zinder, John C.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Doudna Cate, Jamie H.; Blanchard, Scott C.

    2015-01-01

    Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation. PMID:26224058

  17. Low bacterial community diversity in two introduced aphid pests revealed with 16S rRNA amplicon sequencing

    PubMed Central

    Ortiz-Martínez, Sebastían; Silva, Andrea X.; Lavandero, Blas

    2018-01-01

    Bacterial endosymbionts that produce important phenotypic effects on their hosts are common among plant sap-sucking insects. Aphids have become a model system of insect-symbiont interactions. However, endosymbiont research has focused on a few aphid species, making it necessary to make greater efforts to other aphid species through different regions, in order to have a better understanding of the role of endosymbionts in aphids as a group. Aphid endosymbionts have frequently been studied by PCR-based techniques, using species-specific primers, nevertheless this approach may omit other non-target bacteria cohabiting a particular host species. Advances in high-throughput sequencing technologies are complementing our knowledge of microbial communities by allowing us the study of whole microbiome of different organisms. We used a 16S rRNA amplicon sequencing approach to study the microbiome of aphids in order to describe the bacterial community diversity in introduced populations of the cereal aphids, Sitobion avenae and Rhopalosiphum padi in Chile (South America). An absence of secondary endosymbionts and two common secondary endosymbionts of aphids were found in the aphids R. padi and S. avenae, respectively. Of those endosymbionts, Regiella insecticola was the dominant secondary endosymbiont among the aphid samples. In addition, the presence of a previously unidentified bacterial species closely related to a phytopathogenic Pseudomonad species was detected. We discuss these results in relation to the bacterial endosymbiont diversity found in other regions of the native and introduced range of S. avenae and R. padi. A similar endosymbiont diversity has been reported for both aphid species in their native range. However, variation in the secondary endosymbiont infection could be observed among the introduced and native populations of the aphid S. avenae, indicating that aphid-endosymbiont associations can vary across the geographic range of an aphid species. In

  18. High level bacterial contamination of secondary school students' mobile phones.

    PubMed

    Kõljalg, Siiri; Mändar, Rando; Sõber, Tiina; Rööp, Tiiu; Mändar, Reet

    2017-06-01

    While contamination of mobile phones in the hospital has been found to be common in several studies, little information about bacterial abundance on phones used in the community is available. Our aim was to quantitatively determine the bacterial contamination of secondary school students' mobile phones. Altogether 27 mobile phones were studied. The contact plate method and microbial identification using MALDI-TOF mass spectrometer were used for culture studies. Quantitative PCR reaction for detection of universal 16S rRNA, Enterococcus faecalis 16S rRNA and Escherichia coli allantoin permease were performed, and the presence of tetracycline ( tet A, tet B, tet M), erythromycin ( erm B) and sulphonamide ( sul 1) resistance genes was assessed. We found a high median bacterial count on secondary school students' mobile phones (10.5 CFU/cm 2 ) and a median of 17,032 bacterial 16S rRNA gene copies per phone. Potentially pathogenic microbes ( Staphylococcus aureus , Acinetobacter spp. , Pseudomonas spp., Bacillus cereus and Neisseria flavescens ) were found among dominant microbes more often on phones with higher percentage of E. faecalis in total bacterial 16S rRNA. No differences in contamination level or dominating bacterial species between phone owner's gender and between phone types (touch screen/keypad) were found. No antibiotic resistance genes were detected on mobile phone surfaces. Quantitative study methods revealed high level bacterial contamination of secondary school students' mobile phones.

  19. Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

    USDA-ARS?s Scientific Manuscript database

    The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

  20. Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

    PubMed Central

    Schedlbauer, Andreas; Kaminishi, Tatsuya; Ochoa-Lizarralde, Borja; Dhimole, Neha; Zhou, Shu; López-Alonso, Jorge P.

    2015-01-01

    Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins. PMID:25753625

  1. Horizon-Specific Bacterial Community Composition of German Grassland Soils, as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes ▿ †

    PubMed Central

    Will, Christiane; Thürmer, Andrea; Wollherr, Antje; Nacke, Heiko; Herold, Nadine; Schrumpf, Marion; Gutknecht, Jessica; Wubet, Tesfaye; Buscot, François; Daniel, Rolf

    2010-01-01

    The diversity of bacteria in soil is enormous, and soil bacterial communities can vary greatly in structure. Here, we employed a pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to characterize the overall and horizon-specific (A and B horizons) bacterial community compositions in nine grassland soils, which covered three different land use types. The entire data set comprised 752,838 sequences, 600,544 of which could be classified below the domain level. The average number of sequences per horizon was 41,824. The dominant taxonomic groups present in all samples and horizons were the Acidobacteria, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, Firmicutes, and Bacteroidetes. Despite these overarching dominant taxa, the abundance, diversity, and composition of bacterial communities were horizon specific. In almost all cases, the estimated bacterial diversity (H′) was higher in the A horizons than in the corresponding B horizons. In addition, the H′ was positively correlated with the organic carbon content, the total nitrogen content, and the C-to-N ratio, which decreased with soil depth. It appeared that lower land use intensity results in higher bacterial diversity. The majority of sequences affiliated with the Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Spirochaetes, Verrucomicrobia, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria were derived from A horizons, whereas the majority of the sequences related to Acidobacteria, Chloroflexi, Gemmatimonadetes, Nitrospira, TM7, and WS3 originated from B horizons. The distribution of some bacterial phylogenetic groups and subgroups in the different horizons correlated with soil properties such as organic carbon content, total nitrogen content, or microbial biomass. PMID:20729324

  2. Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

    PubMed

    Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

    1995-12-01

    gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct

  3. Impact of protists on a hydrocarbon-degrading bacterial community from deep-sea Gulf of Mexico sediments: A microcosm study

    NASA Astrophysics Data System (ADS)

    Beaudoin, David J.; Carmichael, Catherine A.; Nelson, Robert K.; Reddy, Christopher M.; Teske, Andreas P.; Edgcomb, Virginia P.

    2016-07-01

    In spite of significant advancements towards understanding the dynamics of petroleum hydrocarbon degrading microbial consortia, the impacts (direct or indirect via grazing activities) of bacterivorous protists remain largely unknown. Microcosm experiments were used to examine whether protistan grazing affects the petroleum hydrocarbon degradation capacity of a deep-sea sediment microbial community from an active Gulf of Mexico cold seep. Differences in n-alkane content between native sediment microcosms and those treated with inhibitors of eukaryotes were assessed by comprehensive two-dimensional gas chromatography following 30-90 day incubations and analysis of shifts in microbial community composition using small subunit ribosomal RNA gene clone libraries. More biodegradation was observed in microcosms supplemented with eukaryotic inhibitors. SSU rRNA gene clone libraries from oil-amended treatments revealed an increase in the number of proteobacterial clones (particularly γ-proteobacteria) after spiking sediments with diesel oil. Bacterial community composition shifted, and degradation rates increased, in treatments where protists were inhibited, suggesting protists affect the hydrocarbon degrading capacity of microbial communities in sediments collected at this Gulf of Mexico site.

  4. Further consideration of the phylogeny of some "traditional" heterotrichs (Protista, Ciliophora) of uncertain affinities, based on new sequences of the small subunit rRNA gene.

    PubMed

    Miao, Miao; Song, Weibo; Clamp, John C; Al-Rasheid, Khaled A S; Al-Khedhairy, Abdulaziz A; Al-Arifi, Saud

    2009-01-01

    The systematic relationships and taxonomic positions of the traditional heterotrich genera Condylostentor, Climacostomum, Fabrea, Folliculina, Peritromus, and Condylostoma, as well as the licnophorid genus Licnophora, were re-examined using new data from sequences of the gene coding for small subunit ribosomal RNA. Trees constructed using distance-matrix, Bayesian inference, and maximum-parsimony methods all showed the following relationships: (1) the "traditional" heterotrichs consist of several paraphyletic groups, including the current classes Heterotrichea, Armophorea and part of the Spirotrichea; (2) the class Heterotrichea was confirmed as a monophyletic assemblage based on our analyses of 31 taxa, and the genus Peritromus was demonstrated to be a peripheral group; (3) the genus Licnophora occupied an isolated branch on one side of the deepest divergence in the subphylum Intramacronucleata and was closely affiliated with spirotrichs, armophoreans, and clevelandellids; (4) Condylostentor, a recently defined genus with several truly unique morphological features, is more closely related to Condylostoma than to Stentor; (5) Folliculina, Eufolliculina, and Maristentor always clustered together with high bootstrap support; and (6) Climacostomum occupied a paraphyletic position distant from Fabrea, showing a close relationship with Condylostomatidae and Chattonidiidae despite of modest support.

  5. Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

    PubMed

    Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

    2014-08-15

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  6. Analysis of early bacterial communities on volcanic deposits on the island of Miyake (Miyake-jima), Japan: a 6-year study at a fixed site.

    PubMed

    Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Nanba, Kenji; Oshima, Kenshiro; Hattori, Masahira; Kamijo, Takashi; Ohta, Hiroyuki

    2012-01-01

    Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0-3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)(-1) of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit.

  7. Analysis of Early Bacterial Communities on Volcanic Deposits on the Island of Miyake (Miyake-jima), Japan: a 6-year Study at a Fixed Site

    PubMed Central

    Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Nanba, Kenji; Oshima, Kenshiro; Hattori, Masahira; Kamijo, Takashi; Ohta, Hiroyuki

    2012-01-01

    Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0–3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)−1 of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit. PMID:22075623

  8. Pole-to-pole biogeography of surface and deep marine bacterial communities

    PubMed Central

    Ghiglione, Jean-François; Galand, Pierre E.; Pommier, Thomas; Pedrós-Alió, Carlos; Maas, Elizabeth W.; Bakker, Kevin; Bertilson, Stefan; Kirchman, David L.; Lovejoy, Connie; Yager, Patricia L.; Murray, Alison E.

    2012-01-01

    The Antarctic and Arctic regions offer a unique opportunity to test factors shaping biogeography of marine microbial communities because these regions are geographically far apart, yet share similar selection pressures. Here, we report a comprehensive comparison of bacterioplankton diversity between polar oceans, using standardized methods for pyrosequencing the V6 region of the small subunit ribosomal (SSU) rRNA gene. Bacterial communities from lower latitude oceans were included, providing a global perspective. A clear difference between Southern and Arctic Ocean surface communities was evident, with 78% of operational taxonomic units (OTUs) unique to the Southern Ocean and 70% unique to the Arctic Ocean. Although polar ocean bacterial communities were more similar to each other than to lower latitude pelagic communities, analyses of depths, seasons, and coastal vs. open waters, the Southern and Arctic Ocean bacterioplankton communities consistently clustered separately from each other. Coastal surface Southern and Arctic Ocean communities were more dissimilar from their respective open ocean communities. In contrast, deep ocean communities differed less between poles and lower latitude deep waters and displayed different diversity patterns compared with the surface. In addition, estimated diversity (Chao1) for surface and deep communities did not correlate significantly with latitude or temperature. Our results suggest differences in environmental conditions at the poles and different selection mechanisms controlling surface and deep ocean community structure and diversity. Surface bacterioplankton may be subjected to more short-term, variable conditions, whereas deep communities appear to be structured by longer water-mass residence time and connectivity through ocean circulation. PMID:23045668

  9. High level bacterial contamination of secondary school students’ mobile phones

    PubMed Central

    Kõljalg, Siiri; Mändar, Rando; Sõber, Tiina; Rööp, Tiiu; Mändar, Reet

    2017-01-01

    Introduction While contamination of mobile phones in the hospital has been found to be common in several studies, little information about bacterial abundance on phones used in the community is available. Our aim was to quantitatively determine the bacterial contamination of secondary school students’ mobile phones. Methods Altogether 27 mobile phones were studied. The contact plate method and microbial identification using MALDI-TOF mass spectrometer were used for culture studies. Quantitative PCR reaction for detection of universal 16S rRNA, Enterococcus faecalis 16S rRNA and Escherichia coli allantoin permease were performed, and the presence of tetracycline (tetA, tetB, tetM), erythromycin (ermB) and sulphonamide (sul1) resistance genes was assessed. Results We found a high median bacterial count on secondary school students’ mobile phones (10.5 CFU/cm2) and a median of 17,032 bacterial 16S rRNA gene copies per phone. Potentially pathogenic microbes (Staphylococcus aureus, Acinetobacter spp., Pseudomonas spp., Bacillus cereus and Neisseria flavescens) were found among dominant microbes more often on phones with higher percentage of E. faecalis in total bacterial 16S rRNA. No differences in contamination level or dominating bacterial species between phone owner’s gender and between phone types (touch screen/keypad) were found. No antibiotic resistance genes were detected on mobile phone surfaces. Conclusion Quantitative study methods revealed high level bacterial contamination of secondary school students’ mobile phones. PMID:28626737

  10. Common bacterial responses in six ecosystems exposed to 10 years of elevated atmospheric carbon dioxide.

    PubMed

    Dunbar, John; Eichorst, Stephanie A; Gallegos-Graves, La Verne; Silva, Shannon; Xie, Gary; Hengartner, N W; Evans, R David; Hungate, Bruce A; Jackson, Robert B; Megonigal, J Patrick; Schadt, Christopher W; Vilgalys, Rytas; Zak, Donald R; Kuske, Cheryl R

    2012-05-01

    Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys. Representative bacterial 16S rRNA gene clone sequences were deposited in GenBank with Accession No. JQ366086–JQ387568. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

  11. Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments

    PubMed Central

    Stamper, David M.; Walch, Marianne; Jacobs, Rachel N.

    2003-01-01

    The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD5), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

  12. Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments.

    PubMed

    Stamper, David M; Walch, Marianne; Jacobs, Rachel N

    2003-02-01

    The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.

  13. Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

    PubMed Central

    Marino, Marilena; Innocente, Nadia; Maifreni, Michela; Mounier, Jérôme; Cobo-Díaz, José F.; Coton, Emmanuel; Carraro, Lisa; Cardazzo, Barbara

    2017-01-01

    This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines. PMID:29163411

  14. The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science.

    PubMed

    Ames, Nancy J; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R

    As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

  15. The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science

    PubMed Central

    Ames, Nancy J.; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R.

    2017-01-01

    Background As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and health care practitioners to analyze these microbial communities and their role in health and disease.16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. Objectives The objectives of this review are to: (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Discussion Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists—individuals uniquely positioned to utilize these techniques in future studies in clinical settings. PMID:28252578

  16. A comparative study of COI and 16 S rRNA genes for DNA barcoding of cultivable carps in India.

    PubMed

    Mohanty, Mausumee; Jayasankar, Pallipuram; Sahoo, Lakshman; Das, Paramananda

    2015-02-01

    The 5' region of the mitochondrial DNA gene cytochrome c oxidase subunit I (COI) is the standard marker for DNA barcoding. However, 16 S rRNA has also been advocated for DNA barcoding in many animal species. Herein, we directly compare the usefulness of COI and 16 S rRNA in discriminating six cultivable carp species: Labeo rohita, Catla catla, Cirrhinus mrigala, Labeo fimbriatus, Labeo bata and Cirrhinus reba from India. Analysis of partial sequences of these two gene fragments from 171 individuals indicated close genetic relationship between Catla catla and Labeo rohita. The results of the present study indicated COI to be more useful than 16 S rRNA for DNA barcoding of Indian carps.

  17. Assembly Architecture and DNA Binding of the Bacteriophage P22 Terminase Small Subunit

    PubMed Central

    Němeček, Daniel; Lander, Gabriel C.; Johnson, John E.; Casjens, Sherwood R.; Thomas, George J.

    2008-01-01

    Summary Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally-encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wildtype gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA – involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds dsDNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold, despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation. PMID:18775728

  18. Microbial diversity in the floral nectar of seven Epipactis (Orchidaceae) species

    PubMed Central

    Jacquemyn, Hans; Lenaerts, Marijke; Tyteca, Daniel; Lievens, Bart

    2013-01-01

    Abstract Floral nectar of animal-pollinated plants is commonly infested with microorganisms, yet little is known about the microorganisms inhabiting the floral nectar of orchids. In this study, we investigated microbial communities occurring in the floral nectar of seven Epipactis (Orchidaceae) species. Culturable bacteria and yeasts were isolated and identified by partially sequencing the small subunit (SSU) ribosomal RNA (rRNA) gene and the D1/D2 domains of the large subunit (LSU) rRNA gene, respectively. Using three different culture media, we found that bacteria were common inhabitants of the floral nectar of Epipactis. The most widely distributed bacterial operational taxonomic units (OTUs) in nectar of Epipactis were representatives of the family of Enterobacteriaceae, with an unspecified Enterobacteriaceae bacterium as the most common. In contrast to previous studies investigating microbial communities in floral nectar, very few yeast species (mainly of the genus Cryptococcus) were observed, and most of them occurred in very low densities. Total OTU richness (i.e., the number of bacterial and yeast OTUs per orchid species) varied between 4 and 20. Cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species. This study extends previous efforts to identify microbial communities in floral nectar and indicates that the floral nectar of the orchids investigated mainly contained bacterial communities with moderate phylogenetic diversity. PMID:23836678

  19. Ecophysiology of the developing total bacterial and lactobacillus communities in the terminal small intestine of weaning piglets.

    PubMed

    Pieper, Robert; Janczyk, Pawel; Zeyner, Annette; Smidt, Hauke; Guiard, Volker; Souffrant, Wolfgang Bernhard

    2008-10-01

    Weaning of the pig is generally regarded as a stressful event which could lead to clinical implications because of the changes in the intestinal ecosystem. The functional properties of microbiota inhabiting the pig's small intestine (SI), including lactobacilli which are assumed to exert health-promoting properties, are yet poorly described. Thus, we determined the ecophysiology of bacterial groups and within genus Lactobacillus in the SI of weaning piglets and the impact of dietary changes. The SI contents of 20 piglets, 4 killed at weaning (only sow milk and no creep feed) and 4 killed at 1, 2, 5, and 11 days post weaning (pw; cereal-based diet) were examined for bacterial cell count and bacterial metabolites by fluorescence in situ hybridization (FISH). Lactobacilli were the predominant group in the SI except at 1 day pw because of a marked reduction in their number. On day 11 pw, bifidobacteria and E. coli were not detected, and Enterobacteriaceae and members of the Clostridium coccoides/Eubacterium rectale cluster were only found occasionally. L. sobrius/L. amylovorus became dominant species whereas the abundance of L. salivarius and L. gasseri/johnsonii declined. Concentration of lactic acid increased pw whereas pH, volatile fatty acids, and ammonia decreased. Carbohydrate utilization of 76 Lactobacillus spp. isolates was studied revealing a shift from lactose and galactose to starch, cellobiose, and xylose, suggesting that the bacteria colonizing the SI of piglets adapt to the newly introduced nutrients during the early weaning period. Identification of isolates based on partial 16S rRNA gene sequence data and comparison with fermentation data furthermore suggested adaptation processes below the species level. The results of our study will help to understand intestinal bacterial ecophysiology and to develop nutritional regimes to prevent or counteract complications during the weaning transition.

  20. Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

    PubMed Central

    O’Farrell, Katrina A.; Janssen, Peter H.

    1999-01-01

    Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil. PMID:10473454

  1. Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

    PubMed Central

    Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

    1999-01-01

    The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

  2. Bacterial communities in the phylloplane of Prunus species.

    PubMed

    Jo, Yeonhwa; Cho, Jin Kyong; Choi, Hoseong; Chu, Hyosub; Lian, Sen; Cho, Won Kyong

    2015-04-01

    Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov.

    USDA-ARS?s Scientific Manuscript database

    Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation...

  4. Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

    PubMed Central

    Kitahara, Kei; Kajiura, Akimasa; Sato, Neuza Satomi; Suzuki, Tsutomu

    2007-01-01

    Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function. PMID:17553838

  5. Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

    PubMed

    Pilger, Beatrice D; Cui, Can; Coen, Donald M

    2004-05-01

    The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

  6. Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

    PubMed Central

    Tunney, Michael M.; Patrick, Sheila; Curran, Martin D.; Ramage, Gordon; Hanna, Donna; Nixon, James R.; Gorman, Sean P.; Davis, Richard I.; Anderson, Neil

    1999-01-01

    In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results. PMID:10488193

  7. Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland.

    PubMed

    Schmidt, Mariane; Priemé, Anders; Stougaard, Peter

    2006-12-01

    Bacterial diversity in alkaline (pH 10.4) and permanently cold (4 degrees C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was shown that the majority were cold-active alkaliphiles. T-RFLP analysis revealed distinct bacterial communities in different fractions of three ikaite columns, and, along with sequence analysis, it showed the presence of rich and diverse bacterial communities. Rarefaction analysis showed that the 109 sequenced clones in the 16S rRNA gene library represented between 25 and 65% of the predicted species richness in the three ikaite columns investigated. Phylogenetic analysis of the 16S rRNA gene sequences revealed many sequences with similarity to alkaliphilic or psychrophilic bacteria, and showed that 33% of the cloned sequences and 33% of the cultured bacteria showed less than 97% sequence identity to known sequences in databases, and may therefore represent yet unknown species.

  8. Bacterial Diversity and Nitrogen Utilization Strategies in the Upper Layer of the Northwestern Pacific Ocean.

    PubMed

    Li, Yuan-Yuan; Chen, Xiao-Huang; Xie, Zhang-Xian; Li, Dong-Xu; Wu, Peng-Fei; Kong, Ling-Fen; Lin, Lin; Kao, Shuh-Ji; Wang, Da-Zhi

    2018-01-01

    Nitrogen (N) is a primary limiting nutrient for bacterial growth and productivity in the ocean. To better understand bacterial community and their N utilization strategy in different N regimes of the ocean, we examined bacterial diversity, diazotrophic diversity, and N utilization gene expressions in the northwestern Pacific Ocean (NWPO) using a combination of high-throughput sequencing and real-time qPCR methods. 521 and 204 different operational taxonomic units (OTUs) were identified in the 16s rRNA and nifH libraries from nine surface samples. Of the 16s rRNA gene OTUs, 11.9% were observed in all samples while 3.5 and 15.9% were detected only in N-sufficient and N-deficient samples. Proteobacteria, Cyanobacteria and Bacteroidetes dominated the bacterial community. Prochlorococcus and Pseudoalteromonas were the most abundant at the genus level in N-deficient regimes, while SAR86, Synechococcus and SAR92 were predominant in the Kuroshio-Oyashio confluence region. The distribution of the nifH gene presented great divergence among sampling stations: Cyanobacterium_UCYN-A dominated the N-deficient stations, while clusters related to the Alpha-, Beta- , and Gamma-Proteobacteria were abundant in other stations. Temperature was the main factor that determined bacterial community structure and diversity while concentration of NO X -N was significantly correlated with structure and distribution of N 2 -fixing microorganisms. Expression of the ammonium transporter was much higher than that of urea transporter subunit A ( urtA ) and ferredoxin-nitrate reductase , while urtA had an increased expression in N-deficient surface water. The predicted ammonium transporter and ammonium assimilation enzymes were most abundant in surface samples while urease and nitrogenase were more abundant in the N-deficient regions. These findings underscore the fact that marine bacteria have evolved diverse N utilization strategies to adapt to different N habitats, and that urea metabolism is of

  9. Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus

    PubMed Central

    Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

    2014-01-01

    Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris–HCl pH 8.5. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å. PMID:24419623

  10. Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

    PubMed

    Lake, Nicole J; Webb, Bryn D; Stroud, David A; Richman, Tara R; Ruzzenente, Benedetta; Compton, Alison G; Mountford, Hayley S; Pulman, Juliette; Zangarelli, Coralie; Rio, Marlene; Boddaert, Nathalie; Assouline, Zahra; Sherpa, Mingma D; Schadt, Eric E; Houten, Sander M; Byrnes, James; McCormick, Elizabeth M; Zolkipli-Cunningham, Zarazuela; Haude, Katrina; Zhang, Zhancheng; Retterer, Kyle; Bai, Renkui; Calvo, Sarah E; Mootha, Vamsi K; Christodoulou, John; Rötig, Agnes; Filipovska, Aleksandra; Cristian, Ingrid; Falk, Marni J; Metodiev, Metodi D; Thorburn, David R

    2017-08-03

    The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32 ∗ ]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  11. Nucleolar TRF2 attenuated nucleolus stress-induced HCC cell-cycle arrest by altering rRNA synthesis.

    PubMed

    Yuan, Fuwen; Xu, Chenzhong; Li, Guodong; Tong, Tanjun

    2018-05-03

    The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.

  12. Bacterial diversity in Fe-rich hydrothermal sediments at two South Tonga Arc submarine volcanoes.

    PubMed

    Forget, N L; Murdock, S A; Juniper, S K

    2010-12-01

    Seafloor iron oxide deposits are a common feature of submarine hydrothermal systems. Morphological study of these deposits has led investigators to suggest a microbiological role in their formation, through the oxidation of reduced Fe in hydrothermal fluids. Fe-oxidizing bacteria, including the recently described Zetaproteobacteria, have been isolated from a few of these deposits but generally little is known about the microbial diversity associated with this habitat. In this study, we characterized bacterial diversity in two Fe oxide samples collected on the seafloor of Volcanoes 1 and 19 on the South Tonga Arc. We were particularly interested in confirming the presence of Zetaproteobacteria at these two sites and in documenting the diversity of groups other than Fe oxidizers. Our results (small subunit rRNA gene sequence data) showed a surprisingly high bacterial diversity, with 150 operational taxonomic units belonging to 19 distinct taxonomic groups. Both samples were dominated by Zetaproteobacteria Fe oxidizers. This group was most abundant at Volcano 1, where sediments were richer in Fe and contained more crystalline forms of Fe oxides. Other groups of bacteria found at these two sites include known S- and a few N-metabolizing bacteria, all ubiquitous in marine environments. The low similarity of our clones with the GenBank database suggests that new species and perhaps new families were recovered. The results of this study suggest that Fe-rich hydrothermal sediments, while dominated by Fe oxidizers, can be exploited by a variety of autotrophic and heterotrophic micro-organisms. © 2010 Blackwell Publishing Ltd.

  13. Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis.

    PubMed

    Chim Chan, On; Casper, Peter; Sha, Li Qing; Feng, Zhi Li; Fu, Yun; Yang, Xiao Dong; Ulrich, Andreas; Zou, Xiao Ming

    2008-06-01

    Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.

  14. Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes.

    PubMed

    Stefanova, Katerina; Tomova, Iva; Tomova, Anna; Radchenkova, Nadja; Atanassov, Ivan; Kambourova, Margarita

    2015-12-01

    Archaeal and bacterial diversity in two Bulgarian hot springs, geographically separated with different tectonic origin and different temperature of water was investigated exploring two genes, 16S rRNA and GH-57. Archaeal diversity was significantly higher in the hotter spring Levunovo (LV) (82°C); on the contrary, bacterial diversity was higher in the spring Vetren Dol (VD) (68°C). The analyzed clones from LV library were referred to twenty eight different sequence types belonging to five archaeal groups from Crenarchaeota and Euryarchaeota. A domination of two groups was observed, Candidate Thaumarchaeota and Methanosarcinales. The majority of the clones from VD were referred to HWCG (Hot Water Crenarchaeotic Group). The formation of a group of thermophiles in the order Methanosarcinales was suggested. Phylogenetic analysis revealed high numbers of novel sequences, more than one third of archaeal and half of the bacterial phylotypes displayed similarity lower than 97% with known ones. The retrieved GH-57 gene sequences showed a complex phylogenic distribution. The main part of the retrieved homologous GH-57 sequences affiliated with bacterial phyla Bacteroidetes, Deltaproteobacteria, Candidate Saccharibacteria and affiliation of almost half of the analyzed sequences is not fully resolved. GH-57 gene analysis allows an increased resolution of the biodiversity assessment and in depth analysis of specific taxonomic groups. [Int Microbiol 18(4):217-223 (2015)]. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  15. Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

    PubMed

    Tian, Yang; Li, Yan Hong

    2017-01-01

    To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits.

    PubMed

    Chakraborty, Saswata; Liu, Runhui; Hayouka, Zvi; Chen, Xinyu; Ehrhardt, Jeffrey; Lu, Qin; Burke, Eileen; Yang, Yiqing; Weisblum, Bernard; Wong, Gerard C L; Masters, Kristyn S; Gellman, Samuel H

    2014-10-15

    Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residues, and most HDP-mimetic polymers have therefore contained binary combinations of hydrophobic and cationic subunits. However, HDP-mimetic polymers rarely duplicate the hydrophobic surface and cationic charge density found among HDPs ( Hu , K. ; et al. Macromolecules 2013 , 46 , 1908 ); the charge and hydrophobicity are generally higher among the polymers. Statistical analysis of HDP sequences ( Wang , G. ; et al. Nucleic Acids Res. 2009 , 37 , D933 ) has revealed that serine (polar but uncharged) is a very common HDP constituent and that glycine is more prevalent among HDPs than among proteins in general. These observations prompted us to prepare and evaluate ternary nylon-3 copolymers that contain a modestly polar but uncharged subunit, either serine-like or glycine-like, along with a hydrophobic subunit and a cationic subunit. Starting from binary hydrophobic-cationic copolymers that were previously shown to be highly active against bacteria but also highly hemolytic, we found that replacing a small proportion of the hydrophobic subunit with either of the polar, uncharged subunits can diminish the hemolytic activity with minimal impact on the antibacterial activity. These results indicate that the incorporation of polar, uncharged subunits may be generally useful for optimizing the biological activity profiles of antimicrobial polymers. In the context of HDP evolution, our findings suggest that there is a selective advantage to retaining polar, uncharged residues in natural antimicrobial peptides.

  17. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    DOE PAGES

    Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; ...

    2016-06-02

    We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungalmore » community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.« less

  18. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian

    We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungalmore » community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.« less

  19. Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

    PubMed Central

    Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; Tiedje, James M.

    2016-01-01

    We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation. PMID:27313569

  20. Abundance and activity of 16S rRNA, amoA and nifH bacterial genes during assisted phytostabilization of mine tailings

    PubMed Central

    Nelson, Karis N.; Neilson, Julia W.; Root, Robert A.; Chorover, Jon; Maier, Raina M.

    2014-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nifH, and amoA were monitored during a nine month phytostabilization study using buffalo grass and quailbush grown in compost-amended, metalliferous tailings. The compost amendment provided a greater than 5-log increase in bacterial abundance, and survival of this compost-inoculum was more stable in planted treatments. Despite increased abundance, the activity of the introduced community was low, and significant increases were not detected until six and nine months in quailbush, and unplanted compost and buffalo grass treatments, respectively. In addition, increased abundances of nitrogen-fixation (nifH) and ammonia-oxidizing (amoA) genes were observed in rhizospheres of buffalo grass and quailbush, respectively. Thus, plant establishment facilitated the short term stabilization of introduced bacterial biomass and supported the growth of two key nitrogen-cycling populations in compost-amended tailings. PMID:25495940

  1. Abundance and Activity of 16S rRNA, AmoA and NifH Bacterial Genes During Assisted Phytostabilization of Mine Tailings.

    PubMed

    Nelson, Karis N; Neilson, Julia W; Root, Robert A; Chorover, Jon; Maier, Raina M

    2015-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nifH, and amoA were monitored during a nine month phytostabilization study using buffalo grass and quailbush grown in compost-amended, metalliferous tailings. The compost amendment provided a greater than 5-log increase in bacterial abundance, and survival of this compost-inoculum was more stable in planted treatments. Despite increased abundance, the activity of the introduced community was low, and significant increases were not detected until six and nine months in quailbush, and unplanted compost and buffalo grass treatments, respectively. In addition, increased abundances of nitrogen-fixation (nifH) and ammonia-oxidizing (amoA) genes were observed in rhizospheres of buffalo grass and quailbush, respectively. Thus, plant establishment facilitated the short term stabilization of introduced bacterial biomass and supported the growth of two key nitrogen-cycling populations in compost-amended tailings.

  2. Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

    PubMed Central

    Staley, C.; Sadowsky, M. J.; Gyawali, P.; Sidhu, J. P. S.; Palmer, A.; Beale, D. J.; Toze, S.

    2015-01-01

    In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on

  3. Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

    PubMed

    Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

    2015-10-01

    In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on

  4. Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

    PubMed Central

    Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

    2014-01-01

    SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

  5. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    PubMed Central

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  6. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    PubMed

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  7. Seasonal changes in the digesta-adherent rumen bacterial communities of dairy cattle grazing pasture.

    PubMed

    Noel, Samantha J; Attwood, Graeme T; Rakonjac, Jasna; Moon, Christina D; Waghorn, Garry C; Janssen, Peter H

    2017-01-01

    The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a 'snapshot' in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle.

  8. Seasonal changes in the digesta-adherent rumen bacterial communities of dairy cattle grazing pasture

    PubMed Central

    Attwood, Graeme T.; Rakonjac, Jasna; Moon, Christina D.; Waghorn, Garry C.; Janssen, Peter H.

    2017-01-01

    The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a ‘snapshot’ in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle. PMID:28296930

  9. An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

    PubMed

    Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2016-12-01

    Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Changes in Metabolically Active Bacterial Community during Rumen Development, and Their Alteration by Rhubarb Root Powder Revealed by 16S rRNA Amplicon Sequencing.

    PubMed

    Wang, Zuo; Elekwachi, Chijioke; Jiao, Jinzhen; Wang, Min; Tang, Shaoxun; Zhou, Chuanshe; Tan, Zhiliang; Forster, Robert J

    2017-01-01

    The objective of this present study was to explore the initial establishment of metabolically active bacteria and subsequent evolution in four fractions: rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) through early weaning and supplementing rhubarb root powder in 7 different age groups (1, 10, 20, 38, 41, 50, and 60 d) during rumen development. Results of the 16S rRNA sequencing based on RNA isolated from the four fractions revealed that the potentially active bacterial microbiota in four fractions were dominated by the phyla Proteobacteria, Firmicutes , and Bacteroidetes regardless of different ages. An age-dependent increment of Chao 1 richness was observed in the fractions of RL and RE. The principal coordinate analysis (PCoA) indicated that samples in four fractions all clustered based on different age groups, and the structure of the bacterial community in RE was distinct from those in other three fractions. The abundances of Proteobacteria decreased significantly ( P < 0.05) with age, while increases in the abundances of Firmicutes and Bacteroidetes were noted. At the genus level, the abundance of the predominant genus Mannheimia in the Proteobacteria phylum decreased significantly ( P < 0.05) after 1 d, while the genera Quinella, Prevotella, Fretibacterium, Ruminococcus, Lachnospiraceae NK3A20 group , and Atopobium underwent different manners of increases and dominated the bacterial microbiota across four fractions. Variations of the distributions of some specific bacterial genera across fractions were observed, and supplementation of rhubarb affected the relative abundance of various genera of bacteria.

  11. Changes in Metabolically Active Bacterial Community during Rumen Development, and Their Alteration by Rhubarb Root Powder Revealed by 16S rRNA Amplicon Sequencing

    PubMed Central

    Wang, Zuo; Elekwachi, Chijioke; Jiao, Jinzhen; Wang, Min; Tang, Shaoxun; Zhou, Chuanshe; Tan, Zhiliang; Forster, Robert J.

    2017-01-01

    The objective of this present study was to explore the initial establishment of metabolically active bacteria and subsequent evolution in four fractions: rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) through early weaning and supplementing rhubarb root powder in 7 different age groups (1, 10, 20, 38, 41, 50, and 60 d) during rumen development. Results of the 16S rRNA sequencing based on RNA isolated from the four fractions revealed that the potentially active bacterial microbiota in four fractions were dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes regardless of different ages. An age-dependent increment of Chao 1 richness was observed in the fractions of RL and RE. The principal coordinate analysis (PCoA) indicated that samples in four fractions all clustered based on different age groups, and the structure of the bacterial community in RE was distinct from those in other three fractions. The abundances of Proteobacteria decreased significantly (P < 0.05) with age, while increases in the abundances of Firmicutes and Bacteroidetes were noted. At the genus level, the abundance of the predominant genus Mannheimia in the Proteobacteria phylum decreased significantly (P < 0.05) after 1 d, while the genera Quinella, Prevotella, Fretibacterium, Ruminococcus, Lachnospiraceae NK3A20 group, and Atopobium underwent different manners of increases and dominated the bacterial microbiota across four fractions. Variations of the distributions of some specific bacterial genera across fractions were observed, and supplementation of rhubarb affected the relative abundance of various genera of bacteria. PMID:28223972

  12. High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

    PubMed Central

    Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob; Feld, Louise; Holben, William E.

    2014-01-01

    In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition. PMID:24610853

  13. Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

    PubMed

    Alejo, Jose L; Blanchard, Scott C

    2017-10-10

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

  14. Miscoding-induced stalling of substrate translocation on the bacterial ribosome

    PubMed Central

    Alejo, Jose L.; Blanchard, Scott C.

    2017-01-01

    Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

  15. Genome-Wide Identification and Comprehensive Expression Profiling of Ribosomal Protein Small Subunit (RPS) Genes and their Comparative Analysis with the Large Subunit (RPL) Genes in Rice

    PubMed Central

    Saha, Anusree; Das, Shubhajit; Moin, Mazahar; Dutta, Mouboni; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

    2017-01-01

    Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. Our previous studies on Ribosomal Protein Large subunit (RPL) genes provided insights into their stress responsive roles in rice. In the present study, we have explored the developmental and stress regulated expression patterns of Ribosomal Protein Small (RPS) subunit genes for their differential expression in a spatiotemporal and stress dependent manner. We have also performed an in silico analysis of gene structure, cis-elements in upstream regulatory regions, protein properties and phylogeny. Expression studies of the 34 RPS genes in 13 different tissues of rice covering major growth and developmental stages revealed that their expression was substantially elevated, mostly in shoots and leaves indicating their possible involvement in the development of vegetative organs. The majority of the RPS genes have manifested significant expression under all abiotic stress treatments with ABA, PEG, NaCl, and H2O2. Infection with important rice pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Rhizoctonia solani also induced the up-regulation of several of the RPS genes. RPS4, 13a, 18a, and 4a have shown higher transcript levels under all the abiotic stresses, whereas, RPS4 is up-regulated in both the biotic stress treatments. The information obtained from the present investigation would be useful in appreciating the possible stress-regulatory attributes of the genes coding for rice ribosomal small subunit proteins apart from their functions as house-keeping proteins. A detailed functional analysis of independent genes is required to study their roles in stress tolerance and generating stress- tolerant crops. PMID:28966624

  16. Expression of the ribulose-1,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves

    PubMed Central

    Sheen, Jenq-Yunn; Bogorad, Lawrence

    1986-01-01

    Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for ∼90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute ∼10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:16453739

  17. Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

    PubMed

    Sun, Beili; Zhou, Dongrui; Tu, Jing; Lu, Zuhong

    2017-01-01

    The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

  18. The 11S Proteasome Subunit PSME3 Is a Positive Feedforward Regulator of NF-κB and Important for Host Defense against Bacterial Pathogens.

    PubMed

    Sun, Jinxia; Luan, Yi; Xiang, Dong; Tan, Xiao; Chen, Hui; Deng, Qi; Zhang, Jiaojiao; Chen, Minghui; Huang, Hongjun; Wang, Weichao; Niu, Tingting; Li, Wenjie; Peng, Hu; Li, Shuangxi; Li, Lei; Tang, Wenwen; Li, Xiaotao; Wu, Dianqing; Wang, Ping

    2016-02-02

    The NF-κB pathway plays important roles in immune responses. Although its regulation has been extensively studied, here, we report an unknown feedforward mechanism for the regulation of this pathway by Toll-like receptor (TLR) ligands in macrophages. During bacterial infections, TLR ligands upregulate the expression of the 11S proteasome subunit PSME3 via NF-κB-mediated transcription in macrophages. PSME3, in turn, enhances the transcriptional activity of NF-κB by directly binding to and destabilizing KLF2, a negative regulator of NF-κB transcriptional activity. Consistent with this positive role of PSME3 in NF-κB regulation and importance of the NF-κB pathway in host defense against bacterial infections, the lack of PSME3 in hematopoietic cells renders the hosts more susceptible to bacterial infections, accompanied by increased bacterial burdens in host tissues. Thus, this study identifies a substrate for PSME3 and elucidates a proteolysis-dependent, but ubiquitin-independent, mechanism for NF-κB regulation that is important for host defense and innate immunity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

    PubMed Central

    Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

    2015-01-01

    Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

  20. Characterization of Halophilic Bacterial Communities in Turda Salt Mine (Romania)

    NASA Astrophysics Data System (ADS)

    Carpa, Rahela; Keul, Anca; Muntean, Vasile; Dobrotă, Cristina

    2014-09-01

    Halophilic organisms are having adaptations to extreme salinity, the majority of them being Archaean, which have the ability to grow at extremely high salt concentrations, (from 3 % to 35 %). Level of salinity causes natural fluctuations in the halophilic populations that inhabit this particular habitat, raising problems in maintaining homeostasis of the osmotic pressure. Samples such as salt and water taken from Turda Salt Mine were analyzed in order to identify the eco-physiological bacterial groups. Considering the number of bacteria of each eco-physiological group, the bacterial indicators of salt quality (BISQ) were calculated and studied for each sample. The phosphatase, catalase and dehydrogenases enzymatic activities were quantitatively determined and the enzymatic indicators of salt quality (EISQ) were calculated. Bacterial isolates were analyzed using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene were used. Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level.

  1. Phylogenetic positions of four hypotrichous ciliates (Protista, Ciliophora) based on SSU rRNA gene, with notes on their morphological characters.

    PubMed

    Yang, Caiting; Liu, An; Xu, Yusen; Xu, Yuan; Fan, Xinpeng; Al-Farraj, Saleh A; Ni, Bing; Gu, Fukang

    2015-08-18

     The morphology and infraciliature of the four hypotrichous ciliates; Rigidohymena inquieta (Stokes, 1887) Berger, 2011, Pattersoniella vitiphila Foissner, 1987, Notohymena australis Foissner & O' Donoghue, 1990, and Cyrtohymena (Cyrtohymenides) australis (Foissner, 1995) Foissner, 2004, collected from east China, were investigated by using live observation and protargol impregnation method. An improved diagnosis for R. inquieta was supplied based on descriptions of present and previous populations. New morphology and morphogenesis information based on Chinese populations of another three hypotrichids were also supplemented. The Small-subunit rRNA (SSU rRNA) gene sequences of the four species were characterized and their phylogenetic positions were revealed by means of Bayesian inference and Maximum-likelihood analysis. The analyses shows that R. inquieta clusters with other members of the subfamily Stylonychinae, which confirms the monophyly of the subfamily and verified R. inquieta as a separated species from R. candens though it differs from others mainly by body size. C. (C.) australis occupying the basal position of the clade which contains cyrtohymenids and some other groups, declines the idea of separating Cyrtohymena into two subgenus. Notohymena australis and China population of Pattersoniella vitiphila respectively clustering with their congeners correspond well with the systematics revealed by morphological similarities.

  2. Benthic bacterial diversity in submerged sinkhole ecosystems.

    PubMed

    Nold, Stephen C; Pangborn, Joseph B; Zajack, Heidi A; Kendall, Scott T; Rediske, Richard R; Biddanda, Bopaiah A

    2010-01-01

    Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities.

  3. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota

    PubMed Central

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. PMID:27708630

  4. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota.

    PubMed

    Ellegaard, Kirsten M; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities.

  5. Bacterial communities in soil samples from the Mingyong Glacier of southwestern China.

    PubMed

    Li, Haoyu; Taj, Muhammad Kamran; Ji, Xiuling; Zhang, Qi; Lin, Liangbing; Zhou, Zhimei; Wei, Yunlin

    2017-05-01

    The present study was an effort to determine the bacterial diversity of soils in Mingyong Glacier located at the Meili Snow Mountains of southwestern China. Mingyong Glacier has different climatic zones within a very narrow area, and bacterial community diversity in this low temperature area remains largely unknown. In this study, soil samples were collected from four different climatic zones: M11A (dry warm valley), M14 (forest), M15 (grass land), and M16 (glacier zones). Phylogenetic analysis based on 16S rRNA gene V6 hypervariable region showed high bacterial abundance in the glacier. The number of Operational Taxonomic Units ranged from 2.24×10 3 to 5.56×10 3 in soil samples. Statistical analysis of 16S rRNA gene clone libraries results showed that bacterial diversity in zones M11A,M14 and M16 are higher than in zone M15. The bacterial community structures are clearly distinguishable, and phylogenetic analysis showed that the predominant phyla were Proteobacteria, Deinococcus-Thermus, Firmicutes, Actinobacteria, and Nitrospirae in Mingyong Glacier. Seventy-nine different orders from four zones have been isolated. Bacterial diversity and distribution of bacterial communities related to the anthropogenic perturbations in zone (M15) were confirmed by diversity index analysis, and the diversity index of other three zones was satisfactory through this analysis software. The results suggest that bacterial diversity and distribution analyses using bacterial 16S rRNA gene V6 hypervariable region were successful, and bacterial communities in this area not only had the same bacterial phyla compared to other glaciers but also had their own rare species.

  6. Common and distinguishing features of the bacterial and fungal communities in biological soil crusts and shrub root zone soils

    USGS Publications Warehouse

    Steven, Blaire; Gallegos-Graves, La Verne; Yeager, Chris; Belnap, Jayne; Kuske, Cheryl R.

    2013-01-01

    Soil microbial communities in dryland ecosystems play important roles as root associates of the widely spaced plants and as the dominant members of biological soil crusts (biocrusts) colonizing the plant interspaces. We employed rRNA gene sequencing (bacterial 16S/fungal large subunit) and shotgun metagenomic sequencing to compare the microbial communities inhabiting the root zones of the dominant shrub, Larrea tridentata (creosote bush), and the interspace biocrusts in a Mojave desert shrubland within the Nevada Free Air CO2 Enrichment (FACE) experiment. Most of the numerically abundant bacteria and fungi were present in both the biocrusts and root zones, although the proportional abundance of those members differed significantly between habitats. Biocrust bacteria were predominantly Cyanobacteria while root zones harbored significantly more Actinobacteria and Proteobacteria. Pezizomycetes fungi dominated the biocrusts while Dothideomycetes were highest in root zones. Functional gene abundances in metagenome sequence datasets reflected the taxonomic differences noted in the 16S rRNA datasets. For example, functional categories related to photosynthesis, circadian clock proteins, and heterocyst-associated genes were enriched in the biocrusts, where populations of Cyanobacteria were larger. Genes related to potassium metabolism were also more abundant in the biocrusts, suggesting differences in nutrient cycling between biocrusts and root zones. Finally, ten years of elevated atmospheric CO2 did not result in large shifts in taxonomic composition of the bacterial or fungal communities or the functional gene inventories in the shotgun metagenomes.

  7. Characterization and Evolution of Cell Division and Cell Wall Synthesis Genes in the Bacterial Phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and Phylogenetic Comparison with rRNA Genes▿ †

    PubMed Central

    Pilhofer, Martin; Rappl, Kristina; Eckl, Christina; Bauer, Andreas Peter; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Petroni, Giulio

    2008-01-01

    In the past, studies on the relationships of the bacterial phyla Planctomycetes, Chlamydiae, Lentisphaerae, and Verrucomicrobia using different phylogenetic markers have been controversial. Investigations based on 16S rRNA sequence analyses suggested a relationship of the four phyla, showing the branching order Planctomycetes, Chlamydiae, Verrucomicrobia/Lentisphaerae. Phylogenetic analyses of 23S rRNA genes in this study also support a monophyletic grouping and their branching order—this grouping is significant for understanding cell division, since the major bacterial cell division protein FtsZ is absent from members of two of the phyla Chlamydiae and Planctomycetes. In Verrucomicrobia, knowledge about cell division is mainly restricted to the recent report of ftsZ in the closely related genera Prosthecobacter and Verrucomicrobium. In this study, genes of the conserved division and cell wall (dcw) cluster (ddl, ftsQ, ftsA, and ftsZ) were characterized in all verrucomicrobial subdivisions (1 to 4) with cultivable representatives (1 to 4). Sequence analyses and transcriptional analyses in Verrucomicrobia and genome data analyses in Lentisphaerae suggested that cell division is based on FtsZ in all verrucomicrobial subdivisions and possibly also in the sister phylum Lentisphaerae. Comprehensive sequence analyses of available genome data for representatives of Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes strongly indicate that their last common ancestor possessed a conserved, ancestral type of dcw gene cluster and an FtsZ-based cell division mechanism. This implies that Planctomycetes and Chlamydiae may have shifted independently to a non-FtsZ-based cell division mechanism after their separate branchings from their last common ancestor with Verrucomicrobia. PMID:18310338

  8. Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

    PubMed

    De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

    2002-12-01

    Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.

  9. Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

    PubMed Central

    De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

    2002-01-01

    Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species. PMID:19265950

  10. Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

    PubMed Central

    Klinger, C; Huet, J; Song, D; Petersen, G; Riva, M; Bautz, E K; Sentenac, A; Oudet, P; Schultz, P

    1996-01-01

    Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. Images PMID:8887555

  11. Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

    USDA-ARS?s Scientific Manuscript database

    To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experi...

  12. Buccal Swabbing as a Noninvasive Method To Determine Bacterial, Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

    PubMed Central

    Kirk, Michelle R.; Jonker, Arjan; McCulloch, Alan

    2015-01-01

    Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants. PMID:26276109

  13. Buccal swabbing as a noninvasive method to determine bacterial, archaeal, and eukaryotic microbial community structures in the rumen.

    PubMed

    Kittelmann, Sandra; Kirk, Michelle R; Jonker, Arjan; McCulloch, Alan; Janssen, Peter H

    2015-11-01

    Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas

    PubMed Central

    Meyer, Moritz T.; Genkov, Todor; Skepper, Jeremy N.; Jouhet, Juliette; Mitchell, Madeline C.; Spreitzer, Robert J.; Griffiths, Howard

    2012-01-01

    The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO2-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C4 pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future. PMID:23112177

  15. Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

    PubMed Central

    Ulrich, Nikea; Rosenberger, Abigail; Brislawn, Colin; Wright, Justin; Kessler, Collin; Toole, David; Solomon, Caroline; Strutt, Steven; McClure, Erin

    2016-01-01

    ABSTRACT Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. IMPORTANCE In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study

  16. Bioluminescent symbionts of the Caribbean flashlight fish (Kryptophanaron alfredi) have a single rRNA operon.

    PubMed

    Wolfe, C J; Haygood, M G

    1993-08-01

    Ribosomal RNA (rRNA) operon copy number and gene order were determined for the luminous bacterial symbiont of Kryptophanaron alfredi, an anomalopid (flashlight) fish, and estimated for the luminous symbionts of 3 other fish families and of 3 luminous seawater isolates. Compared with the seawater isolates and other fish symbionts, the copy number of rRNA genes in the K. alfredi symbiont was radically reduced, although gene order appeared conserved among all the strains. The K. alfredi symbiont possesses only a single rRNA operon, whereas the other strains examined have minimum copy numbers ranging from 8 to 11. No difference in copy number was observed between light organ and seawater isolates of the same species, or between isolates of the same species from the light organs of 2 different host families. Thus, the anomalopid symbiosis appears unique among characterized light organ symbioses.

  17. Metamorphosis of a butterfly-associated bacterial community.

    PubMed

    Hammer, Tobin J; McMillan, W Owen; Fierer, Noah

    2014-01-01

    Butterflies are charismatic insects that have long been a focus of biological research. They are also habitats for microorganisms, yet these microbial symbionts are little-studied, despite their likely importance to butterfly ecology and evolution. In particular, the diversity and composition of the microbial communities inhabiting adult butterflies remain uncharacterized, and it is unknown how the larval (caterpillar) and adult microbiota compare. To address these knowledge gaps, we used Illumina sequencing of 16S rRNA genes from internal bacterial communities associated with multiple life stages of the neotropical butterfly Heliconius erato. We found that the leaf-chewing larvae and nectar- and pollen-feeding adults of H. erato contain markedly distinct bacterial communities, a pattern presumably rooted in their distinct diets. Larvae and adult butterflies host relatively small and similar numbers of bacterial phylotypes, but few are common to both stages. The larval microbiota clearly simplifies and reorganizes during metamorphosis; thus, structural changes in a butterfly's bacterial community parallel those in its own morphology. We furthermore identify specific bacterial taxa that may mediate larval and adult feeding biology in Heliconius and other butterflies. Although male and female Heliconius adults differ in reproductive physiology and degree of pollen feeding, bacterial communities associated with H. erato are not sexually dimorphic. Lastly, we show that captive and wild individuals host different microbiota, a finding that may have important implications for the relevance of experimental studies using captive butterflies.

  18. Metamorphosis of a Butterfly-Associated Bacterial Community

    PubMed Central

    Hammer, Tobin J.; McMillan, W. Owen; Fierer, Noah

    2014-01-01

    Butterflies are charismatic insects that have long been a focus of biological research. They are also habitats for microorganisms, yet these microbial symbionts are little-studied, despite their likely importance to butterfly ecology and evolution. In particular, the diversity and composition of the microbial communities inhabiting adult butterflies remain uncharacterized, and it is unknown how the larval (caterpillar) and adult microbiota compare. To address these knowledge gaps, we used Illumina sequencing of 16S rRNA genes from internal bacterial communities associated with multiple life stages of the neotropical butterfly Heliconius erato. We found that the leaf-chewing larvae and nectar- and pollen-feeding adults of H. erato contain markedly distinct bacterial communities, a pattern presumably rooted in their distinct diets. Larvae and adult butterflies host relatively small and similar numbers of bacterial phylotypes, but few are common to both stages. The larval microbiota clearly simplifies and reorganizes during metamorphosis; thus, structural changes in a butterfly's bacterial community parallel those in its own morphology. We furthermore identify specific bacterial taxa that may mediate larval and adult feeding biology in Heliconius and other butterflies. Although male and female Heliconius adults differ in reproductive physiology and degree of pollen feeding, bacterial communities associated with H. erato are not sexually dimorphic. Lastly, we show that captive and wild individuals host different microbiota, a finding that may have important implications for the relevance of experimental studies using captive butterflies. PMID:24466308

  19. Detection of bacterial 16S rRNA using a molecular beacon-based X sensor

    PubMed Central

    Gerasimova, Yulia V.; Kolpashchikov, Dmitry M.

    2012-01-01

    We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a fully complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, E.coli 16S rRNA was detected in real time with the detection limit of ~ 0.17 nM. The high specificity of the analysis was proven by differentiating B.subtilus from E.coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds. PMID:23021850

  20. Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jansson, Janet; Edlund, Anna; Hardeman, Fredrik

    Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities weremore » most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.« less

  1. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    NASA Astrophysics Data System (ADS)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  2. Bacterial diversity, community structure and potential growth rates along an estuarine salinity gradient

    PubMed Central

    Campbell, Barbara J; Kirchman, David L

    2013-01-01

    Very little is known about growth rates of individual bacterial taxa and how they respond to environmental flux. Here, we characterized bacterial community diversity, structure and the relative abundance of 16S rRNA and 16S rRNA genes (rDNA) using pyrosequencing along the salinity gradient in the Delaware Bay. Indices of diversity, evenness, structure and growth rates of the surface bacterial community significantly varied along the transect, reflecting active mixing between the freshwater and marine ends of the estuary. There was no positive correlation between relative abundances of 16S rRNA and rDNA for the entire bacterial community, suggesting that abundance of bacteria does not necessarily reflect potential growth rate or activity. However, for almost half of the individual taxa, 16S rRNA positively correlated with rDNA, suggesting that activity did follow abundance in these cases. The positive relationship between 16S rRNA and rDNA was less in the whole water community than for free-living taxa, indicating that the two communities differed in activity. The 16S rRNA:rDNA ratios of some typically marine taxa reflected differences in light, nutrient concentrations and other environmental factors along the estuarine gradient. The ratios of individual freshwater taxa declined as salinity increased, whereas the 16S rRNA:rDNA ratios of only some typical marine bacteria increased as salinity increased. These data suggest that physical and other bottom-up factors differentially affect growth rates, but not necessarily abundance of individual taxa in this highly variable environment. PMID:22895159

  3. Bacterial community structure in two permafrost wetlands on the Tibetan Plateau and Sanjiang Plain, China.

    PubMed

    Yun, Juanli; Ju, Yiwen; Deng, Yongcui; Zhang, Hongxun

    2014-08-01

    Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10(10) 16S rRNA gene copies per gram of wet soil in both wetlands, with 10(8) pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.

  4. Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty.

    PubMed

    Kalogianni, Despina P; Goura, Sophia; Aletras, Alexios J; Christopoulos, Theodore K; Chanos, Michalis G; Christofidou, Myrto; Skoutelis, Athanasios; Ioannou, Penelope C; Panagiotopoulos, Elias

    2007-02-15

    Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.

  5. Different bacterial communities in ectomycorrhizae and surrounding soil

    PubMed Central

    Vik, Unni; Logares, Ramiro; Blaalid, Rakel; Halvorsen, Rune; Carlsen, Tor; Bakke, Ingrid; Kolstø, Anne-Brit; Økstad, Ole Andreas; Kauserud, Håvard

    2013-01-01

    Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis. PMID:24326907

  6. Activity and bacterial diversity of snow around Russian Antarctic stations.

    PubMed

    Lopatina, Anna; Krylenkov, Vjacheslav; Severinov, Konstantin

    2013-11-01

    The diversity and temporal dynamics of bacterial communities in pristine snow around two Russian Antarctic stations was investigated. Taxonomic analysis of rDNA libraries revealed that snow communities were dominated by bacteria from a small number of operational taxonomic units (OTUs) that underwent dramatic swings in abundance between the 54th (2008-2009) and 55th (2009-2010) Russian Antarctic expeditions. Moreover, analysis of the 55th expedition samples indicated that there was very little, if any, correspondence in abundance of clones belonging to the same OTU present in rDNA and rRNA libraries. The latter result suggests that most rDNA clones originate from bacteria that are not alive and/or active and may have been deposited on the snow surface from the atmosphere. In contrast, clones most abundant in rRNA libraries (mostly belonging to Variovorax, Janthinobacterium, Pseudomonas, and Sphingomonas genera) may be considered as endogenous Antarctic snow inhabitants. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  7. Reassessment of MxiH subunit orientation and fold within native Shigella T3SS needles using surface labelling and solid-state NMR.

    PubMed

    Verasdonck, Joeri; Shen, Da-Kang; Treadgold, Alexander; Arthur, Christopher; Böckmann, Anja; Meier, Beat H; Blocker, Ariel J

    2015-12-01

    T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    PubMed

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  9. Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

    PubMed

    van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

    1991-06-01

    In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

  10. Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass.

    PubMed

    Eichorst, Stephanie A; Joshua, Chijioke; Sathitsuksanoh, Noppadon; Singh, Seema; Simmons, Blake A; Singer, Steven W

    2014-12-01

    Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. [Bacterial diversity within different sections of summer sea-ice samples from the Prydz Bay, Antarctica].

    PubMed

    Ma, Jifei; Du, Zongjun; Luo, Wei; Yu, Yong; Zeng, Yixin; Chen, Bo; Li, Huirong

    2013-02-04

    In order to assess bacterial abundance and diversity within three different sections of summer sea-ice samples collected from the Prydz Bay, Antarctica. Fluorescence in situ hybridization was applied to determine the proportions of Bacteria in sea-ice. Bacterial community composition within sea ice was analyzed by 16S rRNA gene clone library construction. Correlation analysis was performed between the physicochemical parameters and the bacterial diversity and abundance within sea ice. The result of fluorescence in situ hybridization shows that bacteria were abundant in the bottom section, and the concentration of total organic carbon, total organic nitrogen and phosphate may be the main factors for bacterial abundance. In bacterial 16S rRNA gene libraries of sea-ice, nearly complete 16S rRNA gene sequences were grouped into three distinct lineages of Bacteria (gamma-Proteobacteria, alpha-Proteobacteria and Bacteroidetes). Most clone sequences were related to cultured bacterial isolates from the marine environment, arctic and Antarctic sea-ice with high similarity. The member of Bacteroidetes was not detected in the bottom section of sea-ice. The bacterial communities within sea-ice were little heterogeneous at the genus-level between different sections, and the concentration of NH4+ may cause this distribution. The number of bacteria was abundant in the bottom section of sea-ice. Gamma-proteobacteria was the dominant bacterial lineage in sea-ice.

  12. Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

    PubMed

    Ruppitsch, W; Stöger, A; Indra, A; Grif, K; Schabereiter-Gurtner, C; Hirschl, A; Allerberger, F

    2007-03-01

    In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

  13. Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

    PubMed Central

    Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

    2017-01-01

    ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

  14. Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp.

    PubMed

    van Keulen, H; Gutell, R R; Gates, M A; Campbell, S R; Erlandsen, S L; Jarroll, E L; Kulda, J; Meyer, E A

    1993-01-01

    Complete small-subunit rRNA (SSU-rRNA) coding region sequences were determined for two species of the intestinal parasite Giardia: G. ardeae and G. muris, both belonging to the order Diplomonadida, and a free-living member of this order, Hexamita sp. These sequences were compared to published SSU-rDNA sequences from a third member of the genus Giardia, G. duodenalis (often called G. intestinalis or G. lamblia) and various representative organisms from other taxa. Of the three Giardia sequences analyzed, the SSU-rRNA from G. muris is the smallest (1432 bases as compared to 1435 and 1453 for G. ardeae and G. duodenalis, respectively) and has the lowest G+C content (58.9%). The Hexamita SSU-rRNA is the largest in this group, containing 1550 bases. Because the sizes of the SSU-rRNA are prokaryotic rather than typically eukaryotic, the secondary structures of the SSU-rRNAs were constructed. These structures show a number of typically eukaryotic signature sequences. Sequence alignments based on constraints imposed by secondary structure were used for construction of a phylogenetic tree for these four taxa. The results show that of the four diplomonads represented, the Giardia species form a distinct group. The other diplomonad Hexamita and the microsporidium Vairimorpha necatrix appear to be distinct from Giardia.

  15. Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

    PubMed Central

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  16. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    PubMed

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

  17. Thermostable Cross-Protective Subunit Vaccine against Brucella Species

    PubMed Central

    Barabé, Nicole D.; Grigat, Michelle L.; Lee, William E.; Poirier, Robert T.; Jager, Scott J.; Berger, Bradley J.

    2014-01-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 105 CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. PMID:25320267

  18. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Modeling the integration of bacterial rRNA fragments into the human cancer genome.

    PubMed

    Sieber, Karsten B; Gajer, Pawel; Dunning Hotopp, Julie C

    2016-03-21

    Cancer is a disease driven by the accumulation of genomic alterations, including the integration of exogenous DNA into the human somatic genome. We previously identified in silico evidence of DNA fragments from a Pseudomonas-like bacteria integrating into the 5'-UTR of four proto-oncogenes in stomach cancer sequencing data. The functional and biological consequences of these bacterial DNA integrations remain unknown. Modeling of these integrations suggests that the previously identified sequences cover most of the sequence flanking the junction between the bacterial and human DNA. Further examination of these reads reveals that these integrations are rich in guanine nucleotides and the integrated bacterial DNA may have complex transcript secondary structures. The models presented here lay the foundation for future experiments to test if bacterial DNA integrations alter the transcription of the human genes.

  20. Microbial Diversity in Deep-sea Methane Seep Sediments Presented by SSU rRNA Gene Tag Sequencing

    PubMed Central

    Nunoura, Takuro; Takaki, Yoshihiro; Kazama, Hiromi; Hirai, Miho; Ashi, Juichiro; Imachi, Hiroyuki; Takai, Ken

    2012-01-01

    Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth. PMID:22510646

  1. The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.

    2012-03-26

    Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides (1531{sup AUCACCUCC}1539) near the 3{prime} end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530{sup GAUCA}1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530{sup GA}1531 to 1530{sup AG}1531 double mutation severely affects the viability of bacteria. However, whethermore » Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.« less

  2. Benthic Bacterial Diversity in Submerged Sinkhole Ecosystems▿ †

    PubMed Central

    Nold, Stephen C.; Pangborn, Joseph B.; Zajack, Heidi A.; Kendall, Scott T.; Rediske, Richard R.; Biddanda, Bopaiah A.

    2010-01-01

    Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities. PMID:19880643

  3. Energetics of codon-anticodon recognition on the small ribosomal subunit.

    PubMed

    Almlöf, Martin; Andér, Martin; Aqvist, Johan

    2007-01-09

    Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

  4. Phylogenetic Analysis of Myobia musculi (Schranck, 1781) by Using the 18S Small Ribosomal Subunit Sequence

    PubMed Central

    Feldman, Sanford H; Ntenda, Abraham M

    2011-01-01

    We used high-fidelity PCR to amplify 2 overlapping regions of the ribosomal gene complex from the rodent fur mite Myobia musculi. The amplicons encompassed a large portion of the mite's ribosomal gene complex spanning 3128 nucleotides containing the entire 18S rRNA, internal transcribed spacer (ITS) 1, 5.8S rRNA, ITS2, and a portion of the 5′-end of the 28S rRNA. M. musculi’s 179-nucleotide 5.8S rRNA nucleotide sequence was not conserved, so this region was identified by conservation of rRNA secondary structure. Maximum likelihood and Bayesian inference phylogenetic analyses were performed by using multiple sequence alignment consisting of 1524 nucleotides of M. musculi 18S rRNA and homologous sequences from 42 prostigmatid mites and the tick Dermacentor andersoni. The phylograms produced by both methods were in agreement regarding terminal, secondary, and some tertiary phylogenetic relationships among mites. Bayesian inference discriminated most infraordinal relationships between Eleutherengona and Parasitengona mites in the suborder Anystina. Basal relationships between suborders Anystina and Eupodina historically determined by comparing differences in anatomic characteristics were less well-supported by our molecular analysis. Our results recapitulated similar 18S rRNA sequence analyses recently reported. Our study supports M. musculi as belonging to the suborder Anystina, infraorder Eleutherenona, and superfamily Cheyletoidea. PMID:22330574

  5. Diagnosis and management of small intestinal bacterial overgrowth.

    PubMed

    Bohm, Matthew; Siwiec, Robert M; Wo, John M

    2013-06-01

    Small intestinal bacterial overgrowth (SIBO) can result from failure of the gastric acid barrier, failure of small intestinal motility, anatomic alterations, or impairment of systemic and local immunity. The current accepted criteria for the diagnosis of SIBO is the presence of coliform bacteria isolated from the proximal jejunum with >10(5) colony-forming units/mL. A major concern with luminal aspiration is that it is only one random sampling of the small intestine and may not always be representative of the underlying microbiota. A new approach to examine the underlying microbiota uses rapid molecular sequencing, but its clinical utilization is still under active investigation. Clinical manifestations of SIBO are variable and include bloating, flatulence, abdominal distention, abdominal pain, and diarrhea. Severe cases may present with nutrition deficiencies due to malabsorption of micro- and macronutrients. The current management strategies for SIBO center on identifying and correcting underlying causes, addressing nutrition deficiencies, and judicious utilization of antibiotics to treat symptomatic SIBO.

  6. Detection and characterization of Pasteuria 16S rRNA gene sequences from nematodes and soils.

    PubMed

    Duan, Y P; Castro, H F; Hewlett, T E; White, J H; Ogram, A V

    2003-01-01

    Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G + C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100-10,000 Pasteuria endospores (g soil)(-1).

  7. Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

    PubMed

    Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

    2002-04-01

    The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

  8. PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

    PubMed

    Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

    1992-06-01

    Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

  9. Cospeciation in the triplex symbiosis of termite gut protists (Pseudotrichonympha spp.), their hosts, and their bacterial endosymbionts.

    PubMed

    Noda, S; Kitade, O; Inoue, T; Kawai, M; Kanuka, M; Hiroshima, K; Hongoh, Y; Constantino, R; Uys, V; Zhong, J; Kudo, T; Ohkuma, M

    2007-03-01

    A number of cophylogenetic relationships between two organisms namely a host and a symbiont or parasite have been studied to date; however, organismal interactions in nature usually involve multiple members. Here, we investigated the cospeciation of a triplex symbiotic system comprising a hierarchy of three organisms -- termites of the family Rhinotermitidae, cellulolytic protists of the genus Pseudotrichonympha in the guts of these termites, and intracellular bacterial symbionts of the protists. The molecular phylogeny was inferred based on two mitochondrial genes for the termites and nuclear small-subunit rRNA genes for the protists and their endosymbionts, and these were compared. Although intestinal microorganisms are generally considered to have looser associations with the host than intracellular symbionts, the Pseudotrichonympha protists showed almost complete codivergence with the host termites, probably due to strict transmissions by proctodeal trophallaxis or coprophagy based on the social behaviour of the termites. Except for one case, the endosymbiotic bacteria of the protists formed a monophyletic lineage in the order Bacteroidales, and the branching pattern was almost identical to those of the protists and the termites. However, some non-codivergent evolutionary events were evident. The members of this triplex symbiotic system appear to have cospeciated during their evolution with minor exceptions; the evolutionary relationships were probably established by termite sociality and the complex microbial community in the gut.

  10. Rifaximin has minor effects on bacterial composition, inflammation, and bacterial translocation in cirrhosis: A randomized trial.

    PubMed

    Kimer, Nina; Pedersen, Julie S; Tavenier, Juliette; Christensen, Jeffrey E; Busk, Troels M; Hobolth, Lise; Krag, Aleksander; Al-Soud, Waleed Abu; Mortensen, Martin S; Sørensen, Søren J; Møller, Søren; Bendtsen, Flemming

    2018-01-01

    Decompensated cirrhosis is characterized by disturbed hemodynamics, immune dysfunction, and high risk of infections. Translocation of viable bacteria and bacterial products from the gut to the blood is considered a key driver in this process. Intestinal decontamination with rifaximin may reduce bacterial translocation (BT) and decrease inflammation. A randomized, placebo-controlled trial investigated the effects of rifaximin on inflammation and BT in decompensated cirrhosis. Fifty-four out-patients with cirrhosis and ascites were randomized, mean age 56 years (± 8.4), and model for end-stage liver disease score 12 (± 3.9). Patients received rifaximin 550-mg BD (n = 36) or placebo BD (n = 18). Blood and fecal (n = 15) sampling were conducted at baseline and after 4 weeks. Bacterial DNA in blood was determined by real-time qPCR 16S rRNA gene quantification. Bacterial composition in feces was analyzed by 16S rRNA gene sequencing. Circulating markers of inflammation, including tumor necrosis factor alpha, interleukins 6, 10, and 18, stromal cell-derived factor 1-α, transforming growth factor β-1, and high sensitivity C-reactive protein, were unaltered by rifaximin treatment. Rifaximin altered abundance of bacterial taxa in blood marginally, only a decrease in Pseudomonadales was observed. In feces, rifaximin decreased bacterial richness, but effect on particular species was not observed. Subgroup analyses on patients with severely disturbed hemodynamics (n = 34) or activated lipopolysaccharide binding protein (n = 37) revealed no effect of rifaximin. Four weeks of treatment with rifaximin had no impact on the inflammatory state and only minor effects on BT and intestinal bacterial composition in stable, decompensated cirrhosis (NCT01769040). © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  11. Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting

    PubMed Central

    Eichler, Stefan; Christen, Richard; Höltje, Claudia; Westphal, Petra; Bötel, Julia; Brettar, Ingrid; Mehling, Arndt; Höfle, Manfred G.

    2006-01-01

    Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS

  12. Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

    USGS Publications Warehouse

    Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

    2007-01-01

    We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

  13. Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

    NASA Astrophysics Data System (ADS)

    Chen, K.

    2017-01-01

    With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

  14. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

    PubMed Central

    Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Shivaya Valášek, Leoš

    2013-01-01

    There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs. PMID:23804757

  15. Crystallization of the Nonameric Small Terminase Subunit of Bacteriophage P22

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    A Roy; A Bhardwaj; G Cingolani

    2011-12-31

    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometrymore » of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.« less

  16. Crystallization of the Nonameric Small Terminase Subunit of bacteriophage P22

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    A Roy; A Bhardwaj; G Cingoloni

    2011-12-31

    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometrymore » of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.« less

  17. Bacterial community changes in copper and PEX drinking water pipeline biofilms under extra disinfection and magnetic water treatment.

    PubMed

    Inkinen, J; Jayaprakash, B; Ahonen, M; Pitkänen, T; Mäkinen, R; Pursiainen, A; Santo Domingo, J W; Salonen, H; Elk, M; Keinänen-Toivola, M M

    2018-02-01

    To study the stability of biofilms and water quality in pilot scale drinking water copper and PEX pipes in changing conditions (extra disinfection, magnetic water treatment, MWT). Next-generation sequencing (NGS) of 16S ribosomal RNA genes (rDNA) to describe total bacterial community and ribosomal RNA (rRNA) to describe active bacterial members in addition to traditional microbiological methods were applied. Biofilms from control copper and PEX pipes shared same most abundant bacteria (Methylobacterium spp., Sphingomonas spp., Zymomonas spp.) and average species diversities (Shannon 3·8-4·2) in rDNA and rRNA libraries, whereas few of the taxa differed by their abundance such as lower total Mycobacterium spp. occurrence in copper (<0·02%) to PEX (<0·2%) pipes. Extra disinfection (total chlorine increase from c. 0·5 to 1 mg l -1 ) affected total and active population in biofilms seen as decrease in many bacterial species and diversity (Shannon 2·7, P < 0·01, rRNA) and increase in Sphingomonas spp. as compared to control samples. Furthermore, extra-disinfected copper and PEX samples formed separate clusters in unweighted non-metric multidimensional scaling plot (rRNA) similarly to MWT-treated biofilms of copper (but not PEX) pipes that instead showed higher species diversity (Shannon 4·8, P < 0·05 interaction). Minor chlorine dose addition increased selection pressure and many species were sensitive to chlorination. Pipe material seemed to affect mycobacteria occurrence, and bacterial communities with MWT in copper but not in PEX pipes. This study using rRNA showed that chlorination affects especially active fraction of bacterial communities. Copper and PEX differed by the occurrence of some bacterial members despite similar community profiles. © 2017 The Society for Applied Microbiology.

  18. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

    USDA-ARS?s Scientific Manuscript database

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

  19. Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

    PubMed Central

    Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

    2013-01-01

    Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

  20. New record of Apoholosticha sinica (Ciliophora, Urostylida) from the UK: morphology, 18S rRNA gene phylogeny and notes on morphogenesis.

    PubMed

    Hu, Xiaozhong; Fan, Yangbo; Warren, Alan

    2015-08-01

    The benthic urostylid ciliate Apoholosticha sinicaFan et al., 2014 was isolated from a salt marsh at Blakeney, UK, and reinvestigated using light microscopy and small-subunit rRNA gene sequencing. Morphologically, it corresponds well with the original description. Several stages of divisional morphogenesis and physiological reorganization were also observed from which the following could be deduced: (i) the oral apparatus is completely newly built in the proter; (ii) frontal-ventral-transverse cirral anlage II does not produce a buccal cirrus; (iii) each of the posteriormost three or four anlagen contributes one transverse cirrus at its posterior end; (iv) a row of frontoterminal cirri originates from the rearmost frontal-ventral-transverse cirral anlage; (v) the last midventral row is formed from the penultimate frontal-ventral-transverse cirral anlage. Based on new data, two diagnostic features were added to the genus definition: (i) the midventral complex is composed of midventral pairs and midventral row and (ii) pretransverse ventral cirri are absent. Based on a combination of morphological and morphogenetic data, the genus Apoholosticha is assigned to the recently erected subfamily Nothoholostichinae Paiva et al., 2014, which is consistent with sequence comparison and phylogenetic analyses based on SSU rRNA gene data. It is also concluded that this benthic species, previously reported only from China, is not an endemic form.

  1. DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation.

    PubMed

    Tummala, Hemanth; Walne, Amanda J; Williams, Mike; Bockett, Nicholas; Collopy, Laura; Cardoso, Shirleny; Ellison, Alicia; Wynn, Rob; Leblanc, Thierry; Fitzgibbon, Jude; Kelsell, David P; van Heel, David A; Payne, Elspeth; Plagnol, Vincent; Dokal, Inderjeet; Vulliamy, Tom

    2016-07-07

    A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. Copyright © 2016. Published by Elsevier Inc.

  2. The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

    PubMed

    Yang, Guowei; Waterfield, Nicholas R

    2013-01-01

    The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

  3. Potential and use of bacterial small RNAs to combat drug resistance: a systematic review

    PubMed Central

    Liu, Xiaodong; Zhang, Lin; Wong, Sunny Hei; Chan, Matthew TV; Wu, William KK

    2017-01-01

    Background Over the decades, new antibacterial agents have been developed in an attempt to combat drug resistance, but they remain unsuccessful. Recently, a novel class of bacterial gene expression regulators, bacterial small RNAs (sRNAs), has received increasing attention toward their involvement in antibiotic resistance. This systematic review aimed to discuss the potential of these small molecules as antibacterial drug targets. Methods Two investigators performed a comprehensive search of MEDLINE, EmBase, and ISI Web of Knowledge from inception to October 2016, without restriction on language. We included all in vitro and in vivo studies investigating the role of bacterial sRNA in antibiotic resistance. Risk of bias of the included studies was assessed by a modified guideline of Systematic Review Center for Laboratory Animal Experimentation (SYRCLE). Results Initial search yielded 432 articles. After exclusion of non-original articles, 20 were included in this review. Of these, all studies examined bacterial-type strains only. There were neither relevant in vivo nor clinical studies. The SYRCLE scores ranged from to 5 to 7, with an average of 5.9. This implies a moderate risk of bias. sRNAs influenced the antibiotics susceptibility through modulation of gene expression relevant to efflux pumps, cell wall synthesis, and membrane proteins. Conclusion Preclinical studies on bacterial-type strains suggest that modulation of sRNAs could enhance bacterial susceptibility to antibiotics. Further studies on clinical isolates and in vivo models are needed to elucidate the therapeutic value of sRNA modulation on treatment of multidrug-resistant bacterial infection. PMID:29290689

  4. Seasonal changes in bacterial communities associated with healthy and diseased Porites coral in southern Taiwan.

    PubMed

    Lin, Chorng-Horng; Chuang, Chih-Hsiang; Twan, Wen-Hung; Chiou, Shu-Fen; Wong, Tit-Yee; Liu, Jong-Kang; Kao, Chyuan-Yao; Kuo, Jimmy

    2016-12-01

    We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

  5. Modular Assembly of the Bacterial Large Ribosomal Subunit

    PubMed Central

    Davis, Joseph H.; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S.; Lyumkis, Dmitry; Williamson, James R.

    2016-01-01

    SUMMARY The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ~4–5Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be ‘re-routed’ through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. PMID:27912064

  6. Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid

    PubMed Central

    Kotilainen, Pirkko; Jalava, Jari; Meurman, Olli; Lehtonen, Olli-Pekka; Rintala, Esa; Seppälä, Olli-Pekka; Eerola, Erkki; Nikkari, Simo

    1998-01-01

    We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis. PMID:9665992

  7. Subunit mass fingerprinting of mitochondrial complex I.

    PubMed

    Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

    2008-10-01

    We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

  8. Exploiting rRNA operon copy number to investigate bacterial reproductive strategies.

    PubMed

    Roller, Benjamin R K; Stoddard, Steven F; Schmidt, Thomas M

    2016-09-12

    The potential for rapid reproduction is a hallmark of microbial life, but microbes in nature must also survive and compete when growth is constrained by resource availability. Successful reproduction requires different strategies when resources are scarce and when they are abundant 1,2 , but a systematic framework for predicting these reproductive strategies in bacteria has not been available. Here, we show that the number of ribosomal RNA operons (rrn) in bacterial genomes predicts two important components of reproduction-growth rate and growth efficiency-which are favoured under contrasting regimes of resource availability 3,4 . We find that the maximum reproductive rate of bacteria doubles with a doubling of rrn copy number, and the efficiency of carbon use is inversely related to maximal growth rate and rrn copy number. We also identify a feasible explanation for these patterns: the rate and yield of protein synthesis mirror the overall pattern in maximum growth rate and growth efficiency. Furthermore, comparative analysis of genomes from 1,167 bacterial species reveals that rrn copy number predicts traits associated with resource availability, including chemotaxis and genome streamlining. Genome-wide patterns of orthologous gene content covary with rrn copy number, suggesting convergent evolution in response to resource availability. Our findings imply that basic cellular processes adapt in contrasting ways to long-term differences in resource availability. They also establish a basis for predicting changes in bacterial community composition in response to resource perturbations using rrn copy number measurements 5 or inferences 6,7 .

  9. Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

    PubMed Central

    Sharma, Kishor; Tollervey, David

    1999-01-01

    The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0 in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing. PMID:10454548

  10. Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

    PubMed

    Urban, C; Salton, M R

    1983-08-31

    The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

  11. Bacterial community and arsenic functional genes diversity in arsenic contaminated soils from different geographic locations

    PubMed Central

    Gu, Yunfu; D. Van Nostrand, Joy; Wu, Liyou; He, Zhili; Qin, Yujia; Zhao, Fang-Jie; Zhou, Jizhong

    2017-01-01

    To understand how soil microbial communities and arsenic (As) functional genes respond to soil arsenic (As) contamination, five soils contaminated with As at different levels were collected from diverse geographic locations, incubated for 54 days under flooded conditions, and examined by both MiSeq sequencing of 16S rRNA gene amplicons and functional gene microarray (GeoChip 4.0). The results showed that both bacterial community structure and As functional gene structure differed among geographical locations. The diversity of As functional genes correlated positively with the diversity of 16S rRNA genes (P< 0.05). Higher diversities of As functional genes and 16S rRNA genes were observed in the soils with higher available As. Soil pH, phosphate-extractable As, and amorphous Fe content were the most important factors in shaping the bacterial community structure and As transformation functional genes. Geographic location was also important in controlling both the bacterial community and As transformation functional potential. These findings provide insights into the variation of As transformation functional genes in soils contaminated with different levels of As at different geographic locations, and the impact of environmental As contamination on the soil bacterial community. PMID:28475654

  12. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  13. Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

    PubMed Central

    Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

    2014-01-01

    To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

  14. Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure

    PubMed Central

    Wafula, Denis; White, John R.; Canion, Andy; Jagoe, Charles; Pathak, Ashish

    2015-01-01

    Freshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis of nirK, nirS, and nosZ functional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance of Proteobacteria, especially Betaproteobacteria and Gammaproteobacteria; (ii) a decrease in the relative abundance of Actinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in the nirK and nirS denitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria, Acidobacteria, Actinobacteria, Alphaproteobacteria, and the nirS denitrifier guilds were present in the IR pivots than in the NIR plots. Identification of the nir

  15. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

    PubMed

    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  16. Bacterial endophyte communities of three agricultural important grass species differ in their response towards management regimes

    NASA Astrophysics Data System (ADS)

    Wemheuer, Franziska; Kaiser, Kristin; Karlovsky, Petr; Daniel, Rolf; Vidal, Stefan; Wemheuer, Bernd

    2017-01-01

    Endophytic bacteria are critical for plant growth and health. However, compositional and functional responses of bacterial endophyte communities towards agricultural practices are still poorly understood. Hence, we analyzed the influence of fertilizer application and mowing frequency on bacterial endophytes in three agriculturally important grass species. For this purpose, we examined bacterial endophytic communities in aerial plant parts of Dactylis glomerata L., Festuca rubra L., and Lolium perenne L. by pyrotag sequencing of bacterial 16S rRNA genes over two consecutive years. Although management regimes influenced endophyte communities, observed responses were grass species-specific. This might be attributed to several bacteria specifically associated with a single grass species. We further predicted functional profiles from obtained 16S rRNA data. These profiles revealed that predicted abundances of genes involved in plant growth promotion or nitrogen metabolism differed between grass species and between management regimes. Moreover, structural and functional community patterns showed no correlation to each other indicating that plant species-specific selection of endophytes is driven by functional rather than phylogenetic traits. The unique combination of 16S rRNA data and functional profiles provided a holistic picture of compositional and functional responses of bacterial endophytes in agricultural relevant grass species towards management practices.

  17. Ribosomal RNA sequence suggest microsporidia are extremely ancient eukaryotes

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Maddox, J. V.; Friedman, S.; Debrunner-Vossbrinck, B. A.; Woese, C. R.

    1987-01-01

    A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  18. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples.

    PubMed

    Barb, Jennifer J; Oler, Andrew J; Kim, Hyung-Suk; Chalmers, Natalia; Wallen, Gwenyth R; Cashion, Ann; Munson, Peter J; Ames, Nancy J

    2016-01-01

    There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology. This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline. Results are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria. The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points throughout a

  19. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  20. Recognition of Potentially Novel Human Disease-Associated Pathogens by Implementation of Systematic 16S rRNA Gene Sequencing in the Diagnostic Laboratory▿ †

    PubMed Central

    Keller, Peter M.; Rampini, Silvana K.; Büchler, Andrea C.; Eich, Gerhard; Wanner, Roger M.; Speck, Roberto F.; Böttger, Erik C.; Bloemberg, Guido V.

    2010-01-01

    Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease. PMID:20631113

  1. Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

    PubMed Central

    Patarca, R; Dorta, B; Ramirez, J L

    1982-01-01

    As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

  2. Gastrointestinal complaints in runners are not due to small intestinal bacterial overgrowth

    PubMed Central

    2011-01-01

    Background Gastrointestinal complaints are common among long distance runners. We hypothesised that small intestinal bacterial overgrowth (SIBO) is present in long distance runners frequently afflicted with gastrointestinal complaints. Findings Seven long distance runners (5 female, mean age 29.1 years) with gastrointestinal complaints during and immediately after exercise without known gastrointestinal diseases performed Glucose hydrogen breath tests for detection of SIBO one week after a lactose hydrogen breath test checking for lactose intolerance. The most frequent symptoms were diarrhea (5/7, 71%) and flatulence (6/7, 86%). The study was conducted at a laboratory. In none of the subjects a pathological hydrogen production was observed after the intake of glucose. Only in one athlete a pathological hydrogen production was measured after the intake of lactose suggesting lactose intolerance. Conclusions Gastrointestinal disorders in the examined long distance runners were not associated with small intestinal bacterial overgrowth. PMID:21794099

  3. Modular Assembly of the Bacterial Large Ribosomal Subunit.

    PubMed

    Davis, Joseph H; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S; Lyumkis, Dmitry; Williamson, James R

    2016-12-01

    The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae

    PubMed Central

    Schmidt, Christian; Berninghausen, Otto; Becker, Thomas

    2017-01-01

    Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3’end cleavage of the 18S rRNA by Nob1 and how the late factor’s removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit. PMID:29155690

  5. Characterization of bacterial diversity associated with deep sea ferromanganese nodules from the South China Sea.

    PubMed

    Zhang, De-Chao; Liu, Yan-Xia; Li, Xin-Zheng

    2015-09-01

    Deep sea ferromanganese (FeMn) nodules contain metallic mineral resources and have great economic potential. In this study, a combination of culture-dependent and culture-independent (16S rRNA genes clone library and pyrosequencing) methods was used to investigate the bacterial diversity in FeMn nodules from Jiaolong Seamount, the South China Sea. Eleven bacterial strains including some moderate thermophiles were isolated. The majority of strains belonged to the phylum Proteobacteria; one isolate belonged to the phylum Firmicutes. A total of 259 near full-length bacterial 16S rRNA gene sequences in a clone library and 67,079 valid reads obtained using pyrosequencing indicated that members of the Gammaproteobacteria dominated, with the most abundant bacterial genera being Pseudomonas and Alteromonas. Sequence analysis indicated the presence of many organisms whose closest relatives are known manganese oxidizers, iron reducers, hydrogen-oxidizing bacteria and methylotrophs. This is the first reported investigation of bacterial diversity associated with deep sea FeMn nodules from the South China Sea.

  6. The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods

    PubMed Central

    Petti, C. A.; Polage, C. R.; Schreckenberger, P.

    2005-01-01

    Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors. PMID:16333109

  7. Bacterial community changes in an industrial algae production system.

    PubMed

    Fulbright, Scott P; Robbins-Pianka, Adam; Berg-Lyons, Donna; Knight, Rob; Reardon, Kenneth F; Chisholm, Stephen T

    2018-04-01

    While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

  8. Upstream Freshwater and Terrestrial Sources Are Differentially Reflected in the Bacterial Community Structure along a Small Arctic River and Its Estuary

    PubMed Central

    Hauptmann, Aviaja L.; Markussen, Thor N.; Stibal, Marek; Olsen, Nikoline S.; Elberling, Bo; Bælum, Jacob; Sicheritz-Pontén, Thomas; Jacobsen, Carsten S.

    2016-01-01

    Glacier melting and altered precipitation patterns influence Arctic freshwater and coastal ecosystems. Arctic rivers are central to Arctic water ecosystems by linking glacier meltwaters and precipitation with the ocean through transport of particulate matter and microorganisms. However, the impact of different water sources on the microbial communities in Arctic rivers and estuaries remains unknown. In this study we used 16S rRNA gene amplicon sequencing to assess a small river and its estuary on the Disko Island, West Greenland (69°N). Samples were taken in August when there is maximum precipitation and temperatures are high in the Disko Bay area. We describe the bacterial community through a river into the estuary, including communities originating in a glacier and a proglacial lake. Our results show that water from the glacier and lake transports distinct communities into the river in terms of diversity and community composition. Bacteria of terrestrial origin were among the dominating OTUs in the main river, while the glacier and lake supplied the river with water containing fewer terrestrial organisms. Also, more psychrophilic taxa were found in the community supplied by the lake. At the river mouth, the presence of dominant bacterial taxa from the lake and glacier was unnoticeable, but these taxa increased their abundances again further into the estuary. On average 23% of the estuary community consisted of indicator OTUs from different sites along the river. Environmental variables showed only weak correlations with community composition, suggesting that hydrology largely influences the observed patterns. PMID:27708629

  9. Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

    PubMed

    Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

    2016-06-01

    Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied

  10. Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

    PubMed Central

    Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

    2014-01-01

    The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

  11. Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

    PubMed Central

    Steenkamp, D J

    1987-01-01

    The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA. Images Fig. 2. Fig. 3. Fig. 5. PMID:3115254

  12. Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

    PubMed Central

    Gill, Christina; Blow, Frances; Darby, Alistair C.

    2016-01-01

    Background Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. Results After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. Conclusions An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures

  13. Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

    PubMed

    Gill, Christina; van de Wijgert, Janneke H H M; Blow, Frances; Darby, Alistair C

    2016-01-01

    Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies

  14. Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants.

    PubMed

    Alcon-Giner, Cristina; Caim, Shabhonam; Mitra, Suparna; Ketskemety, Jennifer; Wegmann, Udo; Wain, John; Belteki, Gusztav; Clarke, Paul; Hall, Lindsay J

    2017-11-02

    Infants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation. Using ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a 'gold-standard'. Results indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had

  15. CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

    PubMed

    Bai, Baoyan; Moore, Henna M; Laiho, Marikki

    2013-01-01

    CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

  16. The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

    PubMed Central

    Tetaud, Emmanuel; Godard, François; Giraud, Marie-France; Ackerman, Sharon H.; di Rago, Jean-Paul

    2014-01-01

    The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1 and the proton translocation channel in F0 for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0 subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0. PMID:24451261

  17. Cyanobacterial endobionts within a major marine planktonic calcifier (Globigerina bulloides, Foraminifera) revealed by 16S rRNA metabarcoding

    NASA Astrophysics Data System (ADS)

    Bird, Clare; Darling, Kate F.; Russell, Ann D.; Davis, Catherine V.; Fehrenbacher, Jennifer; Free, Andrew; Wyman, Michael; Ngwenya, Bryne T.

    2017-02-01

    We investigated the possibility of bacterial symbiosis in Globigerina bulloides, a palaeoceanographically important, planktonic foraminifer. This marine protist is commonly used in micropalaeontological investigations of climatically sensitive subpolar and temperate water masses as well as wind-driven upwelling regions of the world's oceans. G. bulloides is unusual because it lacks the protist algal symbionts that are often found in other spinose species. In addition, it has a large offset in its stable carbon and oxygen isotopic compositions compared to other planktonic foraminifer species, and also that predicted from seawater equilibrium. This is suggestive of novel differences in ecology and life history of G. bulloides, making it a good candidate for investigating the potential for bacterial symbiosis as a contributory factor influencing shell calcification. Such information is essential to evaluate fully the potential response of G. bulloides to ocean acidification and climate change. To investigate possible ecological interactions between G. bulloides and marine bacteria, 18S rRNA gene sequencing, fluorescence microscopy, 16S rRNA gene metabarcoding and transmission electron microscopy (TEM) were performed on individual specimens of G. bulloides (type IId) collected from two locations in the California Current. Intracellular DNA extracted from five G. bulloides specimens was subjected to 16S rRNA gene metabarcoding and, remarkably, 37-87 % of all 16S rRNA gene sequences recovered were assigned to operational taxonomic units (OTUs) from the picocyanobacterium Synechococcus. This finding was supported by TEM observations of intact Synechococcus cells in both the cytoplasm and vacuoles of G. bulloides. Their concentrations were up to 4 orders of magnitude greater inside the foraminifera than those reported for the California Current water column and approximately 5 % of the intracellular Synechococcus cells observed were undergoing cell division. This suggests

  18. 5S rRNA and ribosome.

    PubMed

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  19. Deep 16S rRNA Pyrosequencing Reveals a Bacterial Community Associated with Banana Fusarium Wilt Disease Suppression Induced by Bio-Organic Fertilizer Application

    PubMed Central

    Ruan, Yunze; Xue, Chao; Zhang, Jian; Li, Rong; Shen, Qirong

    2014-01-01

    Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas. PMID:24871319

  20. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  1. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  2. Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

    PubMed

    Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

    2003-05-01

    We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

  3. Bacterial diversity associated with the rotifer Brachionus plicatilis sp. complex determined by culture-dependent and -independent methods.

    PubMed

    Ishino, Ryota; Iehata, Shunpei; Nakano, Miyo; Tanaka, Reiji; Yoshimatsu, Takao; Maeda, Hiroto

    2012-03-01

    The bacterial communities associated with rotifers (Brachionus plicatilis sp. complex) and their culture water were determined using culture-dependent and -independent methods (16S rRNA gene clone library). The bacterial communities determined by the culture-independent method were more diverse than those determined by the culture-dependent method. Although the culture-dependent method indicated the bacterial community of rotifers was relatively similar to that of the culture water, 16S rRNA gene clone library analyses revealed a great difference between the two microbiotas. Our results suggest that most bacteria associated with rotifers are not easily cultured using conventional methods, and that the microbiota of rotifers do not correspond with that of the culture water completely.

  4. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  5. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  6. Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

    PubMed

    Harris, J Kirk; Caporaso, J Gregory; Walker, Jeffrey J; Spear, John R; Gold, Nicholas J; Robertson, Charles E; Hugenholtz, Philip; Goodrich, Julia; McDonald, Daniel; Knights, Dan; Marshall, Paul; Tufo, Henry; Knight, Rob; Pace, Norman R

    2013-01-01

    The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

  7. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  8. Diversity and Assembling Processes of Bacterial Communities in Cryoconite Holes of a Karakoram Glacier.

    PubMed

    Ambrosini, Roberto; Musitelli, Federica; Navarra, Federico; Tagliaferri, Ilario; Gandolfi, Isabella; Bestetti, Giuseppina; Mayer, Christoph; Minora, Umberto; Azzoni, Roberto Sergio; Diolaiuti, Guglielmina; Smiraglia, Claudio; Franzetti, Andrea

    2017-05-01

    Cryoconite holes are small ponds that form on the surface of glaciers that contain a dark debris, the cryoconite, at the bottom and host active ecological communities. Differences in the structure of bacterial communities have been documented among Arctic and mountain glaciers, and among glaciers in different areas of the world. In this study, we investigated the structure of bacterial communities of cryoconite holes of Baltoro Glacier, a large (62 km in length and 524 km 2 of surface) glacier of the Karakoram, by high-throughput sequencing of the V5-V6 hypervariable regions of the 16S rRNA gene. We found that Betaproteobacteria dominated bacterial communities, with large abundance of genera Polaromonas, probably thanks to its highly versatile metabolism, and Limnohabitans, which may have been favoured by the presence of supraglacial lakes in the area where cryoconite holes were sampled. Variation in bacterial communities among different sampling areas of the glacier could be explained by divergent selective processes driven by variation in environmental conditions, particularly pH, which was the only environmental variable that significantly affected the structure of bacterial communities. This variability may be due to both temporal and spatial patterns of variation in environmental conditions.

  9. Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

    PubMed

    Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

    2018-03-19

    To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

  10. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles

    PubMed Central

    Caswell, Clayton C.; Oglesby-Sherrouse, Amanda G.; Murphy, Erin R.

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators. PMID:25389522

  11. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

    PubMed

    Caswell, Clayton C; Oglesby-Sherrouse, Amanda G; Murphy, Erin R

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

  12. Identification of an Alternative rRNA Post-transcriptional Maturation of 26S rRNA in the Kingdom Fungi.

    PubMed

    Navarro-Ródenas, Alfonso; Carra, Andrea; Morte, Asunción

    2018-01-01

    Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap . rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.

  13. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  14. Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod

    PubMed Central

    Ngangbam, Ajit Kumar; Baten, Abdul; Waters, Daniel L. E.; Whalan, Steve; Benkendorff, Kirsten

    2015-01-01

    Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita. PMID:26488885

  15. Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

    PubMed Central

    Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

    2007-01-01

    Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

  16. Spatial pattern in Antarctica: what can we learn from Antarctic bacterial isolates?

    PubMed

    Chong, Chun Wie; Goh, Yuh Shan; Convey, Peter; Pearce, David; Tan, Irene Kit Ping

    2013-09-01

    A range of small- to moderate-scale studies of patterns in bacterial biodiversity have been conducted in Antarctica over the last two decades, most suggesting strong correlations between the described bacterial communities and elements of local environmental heterogeneity. However, very few of these studies have advanced interpretations in terms of spatially associated patterns, despite increasing evidence of patterns in bacterial biogeography globally. This is likely to be a consequence of restricted sampling coverage, with most studies to date focusing only on a few localities within a specific Antarctic region. Clearly, there is now a need for synthesis over a much larger spatial to consolidate the available data. In this study, we collated Antarctic bacterial culture identities based on the 16S rRNA gene information available in the literature and the GenBank database (n > 2,000 sequences). In contrast to some recent evidence for a distinct Antarctic microbiome, our phylogenetic comparisons show that a majority (~75 %) of Antarctic bacterial isolates were highly similar (≥99 % sequence similarity) to those retrieved from tropical and temperate regions, suggesting widespread distribution of eurythermal mesophiles in Antarctic environments. However, across different Antarctic regions, the dominant bacterial genera exhibit some spatially distinct diversity patterns analogous to those recently proposed for Antarctic terrestrial macroorganisms. Taken together, our results highlight the threat of cross-regional homogenisation in Antarctic biodiversity, and the imperative to include microbiota within the framework of biosecurity measures for Antarctica.

  17. Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria

    PubMed Central

    Srinivasan, Sujatha; Hoffman, Noah G.; Morgan, Martin T.; Matsen, Frederick A.; Fiedler, Tina L.; Hall, Robert W.; Ross, Frederick J.; McCoy, Connor O.; Bumgarner, Roger; Marrazzo, Jeanne M.; Fredricks, David N.

    2012-01-01

    Background Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV. Methodology/Principal Findings Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel’s clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel’s criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV. Conclusions/Significance The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in

  18. Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem

    PubMed Central

    Tang, Pei-Zhong; Becker, Scott; Hoang, Tony; Bilgin, Damla; Lim, Yan Wei; Peterson, Todd C.; Mayfield, Stephen; Haerizadeh, Farzad; Shurin, Jonathan B.; Bafna, Vineet; McBride, Robert

    2016-01-01

    Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms. PMID:26896141

  19. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

  20. Antarctic ice core samples: culturable bacterial diversity.

    PubMed

    Shivaji, Sisinthy; Begum, Zareena; Shiva Nageswara Rao, Singireesu Soma; Vishnu Vardhan Reddy, Puram V; Manasa, Poorna; Sailaja, Buddi; Prathiba, Mambatta S; Thamban, Meloth; Krishnan, Kottekkatu P; Singh, Shiv M; Srinivas, Tanuku N R

    2013-01-01

    Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 × 10(3) CFU ml(-1) of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2-100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 °C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    PubMed

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. 16S rRNA Gene-Based Metagenomic Analysis of Ozark Cave Bacteria.

    PubMed

    Oliveira, Cássia; Gunderman, Lauren; Coles, Cathryn A; Lochmann, Jason; Parks, Megan; Ballard, Ethan; Glazko, Galina; Rahmatallah, Yasir; Tackett, Alan J; Thomas, David J

    2017-09-01

    The microbial diversity within cave ecosystems is largely unknown. Ozark caves maintain a year-round stable temperature (12-14 °C), but most parts of the caves experience complete darkness. The lack of sunlight and geological isolation from surface-energy inputs generate nutrient-poor conditions that may limit species diversity in such environments. Although microorganisms play a crucial role in sustaining life on Earth and impacting human health, little is known about their diversity, ecology, and evolution in community structures. We used five Ozark region caves as test sites for exploring bacterial diversity and monitoring long-term biodiversity. Illumina MiSeq sequencing of five cave soil samples and a control sample revealed a total of 49 bacterial phyla, with seven major phyla: Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, Chloroflexi, Bacteroidetes, and Nitrospirae. Variation in bacterial composition was observed among the five caves studied. Sandtown Cave had the lowest richness and most divergent community composition. 16S rRNA gene-based metagenomic analysis of cave-dwelling microbial communities in the Ozark caves revealed that species abundance and diversity are vast and included ecologically, agriculturally, and economically relevant taxa.

  3. 16S rRNA Gene-Based Metagenomic Analysis of Ozark Cave Bacteria

    PubMed Central

    Oliveira, Cássia; Gunderman, Lauren; Coles, Cathryn A.; Lochmann, Jason; Parks, Megan; Ballard, Ethan; Glazko, Galina; Rahmatallah, Yasir; Tackett, Alan J.; Thomas, David J.

    2018-01-01

    The microbial diversity within cave ecosystems is largely unknown. Ozark caves maintain a year-round stable temperature (12–14 °C), but most parts of the caves experience complete darkness. The lack of sunlight and geological isolation from surface-energy inputs generate nutrient-poor conditions that may limit species diversity in such environments. Although microorganisms play a crucial role in sustaining life on Earth and impacting human health, little is known about their diversity, ecology, and evolution in community structures. We used five Ozark region caves as test sites for exploring bacterial diversity and monitoring long-term biodiversity. Illumina MiSeq sequencing of five cave soil samples and a control sample revealed a total of 49 bacterial phyla, with seven major phyla: Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, Chloroflexi, Bacteroidetes, and Nitrospirae. Variation in bacterial composition was observed among the five caves studied. Sandtown Cave had the lowest richness and most divergent community composition. 16S rRNA gene-based metagenomic analysis of cave-dwelling microbial communities in the Ozark caves revealed that species abundance and diversity are vast and included ecologically, agriculturally, and economically relevant taxa. PMID:29551950

  4. Bacterial diversity of the Colombian fermented milk "Suero Costeño" assessed by culturing and high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons.

    PubMed

    Motato, Karina Edith; Milani, Christian; Ventura, Marco; Valencia, Francia Elena; Ruas-Madiedo, Patricia; Delgado, Susana

    2017-12-01

    "Suero Costeño" (SC) is a traditional soured cream elaborated from raw milk in the Northern-Caribbean coast of Colombia. The natural microbiota that characterizes this popular Colombian fermented milk is unknown, although several culturing studies have previously been attempted. In this work, the microbiota associated with SC from three manufacturers in two regions, "Planeta Rica" (Córdoba) and "Caucasia" (Antioquia), was analysed by means of culturing methods in combination with high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons. The bacterial ecosystem of SC samples was revealed to be composed of lactic acid bacteria belonging to the Streptococcaceae and Lactobacillaceae families; the proportions and genera varying among manufacturers and region of elaboration. Members of the Lactobacillus acidophilus group, Lactocococcus lactis, Streptococcus infantarius and Streptococcus salivarius characterized this artisanal product. In comparison with culturing, the use of molecular in deep culture-independent techniques provides a more realistic picture of the overall bacterial communities residing in SC. Besides the descriptive purpose, these approaches will facilitate a rational strategy to follow (culture media and growing conditions) for the isolation of indigenous strains that allow standardization in the manufacture of SC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Chronic N-amended soils exhibit an altered bacterial community structure in Harvard Forest, MA, USA

    Treesearch

    Swathi A. Turlapati; Rakesh Minocha; Premsai S. Bhiravarasa; Louise S. Tisa; William K. Thomas; Subhash C. Minocha

    2013-01-01

    At the Harvard Forest, Petersham, MA, the impact of 20 years of annual ammonium nitrate application to the mixed hardwood stand on soil bacterial communities was studied using 16S rRNA genes pyrosequencing. Amplification of 16S rRNA genes was done using DNA extracted from 30 soil samples (three treatments x two horizons x five subplots) collected from untreated (...

  6. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

    PubMed

    Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

    2016-04-21

    Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

  7. Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

    PubMed

    Preveena, Jagadesan; Bhore, Subhash J

    2013-01-01

    In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study.

  8. Comparative molecular analysis of chemolithoautotrophic bacterial diversity and community structure from coastal saline soils, Gujarat, India

    PubMed Central

    2012-01-01

    Background Soils harbour high diversity of obligate as well as facultative chemolithoautotrophic bacteria that contribute significantly to CO2 dynamics in soil. In this study, we used culture dependent and independent methods to assess the community structure and diversity of chemolithoautotrophs in agricultural and coastal barren saline soils (low and high salinity). We studied the composition and distribution of chemolithoautotrophs by means of functional marker gene cbbL encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a phylogenetic marker 16S rRNA gene. The cbbL form IA and IC genes associated with carbon fixation were analyzed to gain insight into metabolic potential of chemolithoautotrophs in three soil types of coastal ecosystems which had a very different salt load and sulphur content. Results In cbbL libraries, the cbbL form IA was retrieved only from high saline soil whereas form IC was found in all three soil types. The form IC cbbL was also amplified from bacterial isolates obtained from all soil types. A number of novel monophyletic lineages affiliated with form IA and IC phylogenetic trees were found. These were distantly related to the known cbbL sequences from agroecosystem, volcanic ashes and marine environments. In 16S rRNA clone libraries, the agricultural soil was dominated by chemolithoautotrophs (Betaproteobacteria) whereas photoautotrophic Chloroflexi and sulphide oxidizers dominated saline ecosystems. Environmental specificity was apparently visible at both higher taxonomic levels (phylum) and lower taxonomic levels (genus and species). The differentiation in community structure and diversity in three soil ecosystems was supported by LIBSHUFF (P = 0.001) and UniFrac. Conclusion This study may provide fundamentally new insights into the role of chemolithoautotrophic and photoautotrophic bacterial diversity in biochemical carbon cycling in barren saline soils. The bacterial communities varied greatly among the

  9. Characterization of bacterial consortia capable of degrading 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions.

    PubMed

    Song, Bongkeun; Kerkhof, Lee J; Häggblom, Max M

    2002-08-06

    4-Chlorobenzoate and 4-bromobenzoate were readily degraded in denitrifying enrichment cultures established with river sediment, estuarine sediment or agricultural soil as inoculum. Stable denitrifying consortia were obtained and maintained by serial dilution and repeated feeding of substrates. Microbial community analyses were performed to characterize the 4-chlorobenzoate and 4-bromobenzoate degrading consortia with terminal restriction fragment length polymorphism (T-RFLP) and cloning of 16S rRNA genes from the cultures. Interestingly, two major terminal restriction fragments (T-RFs) in the 4-chlorobenzoate degrading consortia and one T-RF in the 4-bromobenzoate utilizing consortium were observed from T-RFLP analysis regardless of their geographical and ecological origins. The two T-RFs (clones 4CB1 and 4CB2) in 4-chlorobenzoate degrading consortia were identified as members of the beta-subunit of the Proteobacteria on the basis of 16S rRNA sequencing analysis. Phylogenetic analysis of 16S rRNA genes showed that clone 4CB1 was closely related to Thauera aromatica while clone 4CB2 was distantly related to the genera Limnobacter and Ralstonia. The 4-bromobenzoate utilizing consortium mainly consisted of one T-RF, which was identical to clone 4CB2 in spite of different enrichment substrate. This suggests that degradation of 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions was mediated by bacteria belonging to the beta-subunit of the Proteobacteria.

  10. Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR

    PubMed Central

    Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

    2005-01-01

    The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4°C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 × 105-fold, 1.5 × 105-fold, and 1.0 × 104-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world. PMID:15640179

  11. Seasonal change in bacterial flora and biomass in mountain snow from the Tateyama Mountains, Japan, analyzed by 16S rRNA gene sequencing and real-time PCR.

    PubMed

    Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

    2005-01-01

    The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4 degrees C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 x 10(5)-fold, 1.5 x 10(5)-fold, and 1.0 x 10(4)-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

  12. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed Central

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-01-01

    Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

  13. Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der.

    PubMed

    Hwang, Jihwan; Tseitin, Vladimir; Ramnarayan, Kal; Shenderovich, Mark D; Inouye, Masayori

    2012-05-01

    Der is an essential and widely conserved GTPase that assists assembly of a large ribosomal subunit in bacteria. Der associates specifically with the 50S subunit in a GTP-dependent manner and the cells depleted of Der accumulate the structurally unstable 50S subunit, which dissociates into an aberrant subunit at a lower Mg(2+) concentration. As Der is an essential and ubiquitous protein in bacteria, it may prove to be an ideal cellular target against which new antibiotics can be developed. In the present study, we describe our attempts to identify novel antibiotics specifically targeting Der GTPase. We performed the structure-based design of Der inhibitors using the X-ray crystal structure of Thermotoga maritima Der (TmDer). Virtual screening of commercially available chemical library retrieved 257 small molecules that potentially inhibit Der GTPase activity. These 257 chemicals were tested for their in vitro effects on TmDer GTPase and in vivo antibacterial activities. We identified three structurally diverse compounds, SBI-34462, -34566 and -34612, that are both biologically active against bacterial cells and putative enzymatic inhibitors of Der GTPase homologs. We also presented the possible interactions of each compound with the Der GTP-binding site to understand the mechanism of inhibition. Therefore, our lead compounds inhibiting Der GTPase provide scaffolds for the development of novel antibiotics against antibiotic-resistant pathogenic bacteria.

  14. The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

    PubMed Central

    Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

    2013-01-01

    Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications

  15. Increased 5S rRNA oxidation in Alzheimer's disease.

    PubMed

    Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

    2012-01-01

    It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

  16. Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches.

    PubMed

    Hao, Da Cheng; Ge, Guang Bo; Yang, Ling

    2008-07-01

    The regional variability of Taxus rhizosphere bacterial community composition and diversity was studied by comparative analysis of three large 16S rRNA gene clone libraries from the Taxus rhizosphere in different regions of China (subtropical and temperate regions). One hundred and forty-six clones were screened for three libraries. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the abundance of sequences affiliated with Gammaproteobacteria, Betaproteobacteria, and Actinobacteria was higher in the library from the T. xmedia rhizosphere of the temperate region compared with the subtropical Taxus mairei rhizosphere. On the other hand, Acidobacteria was more abundant in libraries from the subtropical Taxus mairei rhizosphere. Richness estimates and diversity indices of three libraries revealed major differences, indicating a higher richness in the Taxus rhizosphere bacterial communities of the subtropical region and considerable variability in the bacterial community composition within this region. By enrichment culture, a novel Actinobacteria strain DICP16 was isolated from the T. xmedia rhizosphere of the temperate region and was identified as Leifsonia shinshuensis sp. via 16S rRNA gene and gyrase B sequence analyses. DICP16 was able to remove the xylosyl group from 7-xylosyl-10-deacetylbaccatin III and 7-xylosyl-10-deacetylpaclitaxel, thereby making the xylosyltaxanes available as sources of 10-deacetylbaccatin III and the anticancer drug paclitaxel. Taken together, the present studies provide, for the first time, the knowledge of the biodiversity of microorganisms populating Taxus rhizospheres.

  17. [Congenital skull base defect causing recurrent bacterial meningitis].

    PubMed

    Berliner, Elihay; Bar Meir, Maskit; Megged, Orli

    2012-08-01

    Bacterial meningitis is a life threatening disease. Most patients will experience only one episode throughout life. Children who experience bacterial meningitis more than once, require further immunologic or anatomic evaluation. We report a 9 year old child with five episodes of bacterial meningitis due to a congenital defect of the skull base. A two and a half year old boy first presented to our medical center with pneumococcal meningitis. He was treated with antibiotics and fully recovered. Two months later he presented again with a similar clinical picture. Streptococcus pneumoniae grew in cerebrospinal fluid (CSF) culture. CT scan and later MRI of the brain revealed a defect in the anterior middle fossa floor, with protrusion of brain tissue into the sphenoidal sinus. Corrective surgery was recommended but the parents refused. Three months later, a third episode of pneumococcal meningitis occurred. The child again recovered with antibiotics and this time corrective surgery was performed. Five years later, the boy presented once again with clinical signs and symptoms consistent with bacterial meningitis. CSF culture was positive, but the final identification of the bacteria was conducted by broad spectrum 16S ribosomal RNA PCR (16S rRNA PCR) which revealed a sequence of Neisseria lactamica. CT and MRI showed recurrence of the skull base defect with encephalocele in the sphenoid sinus. The parents again refused neurosurgical intervention. A year later the patient presented with bacterial meningitis. CSF culture obtained after initiation of antibiotics was negative, but actinobacillus was identified in the CSF by 16S rRNA PCR. The patient is scheduled for neurosurgical intervention. In patients with recurrent bacterial meningitis caused by organisms colonizing the oropharynx or nasopharynx, an anatomical defect should be carefully sought and surgically repaired.

  18. Ballistosporomyces changbaiensis sp. nov. and Ballistosporomyces bomiensis sp. nov., two novel species isolated from shrub plant leaves.

    PubMed

    Han, Pei-Jie; Li, Ai-Hua; Wang, Qi-Ming; Bai, Feng-Yan

    2016-07-01

    Four strains, CB 266(T), CB 272, XZ 44D1(T) and XZ 49D2, isolated from shrub plant leaves in China were identified as two novel species of the genus Ballistosporomyces by the sequence analysis of the small subunit of ribosomal RNA (SSU rRNA), the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and internal transcribed spacer (ITS) + 5.8S rRNA region, and physiological comparisons. Ballistosporomyces changbaiensis sp. nov. (type strain CB 266(T) = CGMCC 2.02298(T) = CBS 10124(T), Mycobank number MB 815700) and Ballistosporomyces bomiensis sp. nov. (type strain XZ 44D1(T) = CGMCC 2.02661(T) = CBS 12512(T), Mycobank number MB 815701) are proposed to accommodate these two new species.

  19. Bacterial flagellin—a potent immunomodulatory agent

    PubMed Central

    Hajam, Irshad A; Dar, Pervaiz A; Shahnawaz, Imam; Jaume, Juan Carlos; Lee, John Hwa

    2017-01-01

    Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent, and we address important areas of future research interests. PMID:28860663

  20. Phylogenetic relationships of Sarcocystis neurona of horses and opossums to other cyst-forming coccidia deduced from SSU rRNA gene sequences.

    PubMed

    Elsheikha, Hany M; Lacher, David W; Mansfield, Linda S

    2005-11-01

    Phylogenetic analyses based on sequences of the nuclear-encoded small subunit rRNA (ssurRNA) gene were performed to examine the origin, phylogeny, and biogeographic relationships of Sarcocystis neurona isolates from opossums and horses from the State of Michigan, USA, in relation to other cyst-forming coccidia. A total of 31 taxa representing all recognized subfamilies and genera of Sarcocystidae were included in the analyses with clonal isolates of two opossum and two horse S. neurona. Phylogenies obtained by the four tree-building methods were consistent with the classical taxonomy based on morphological criteria. The "isosporid" coccidia Neospora, Toxoplasma, Besnoitia, Isospora lacking stieda bodies, and Hyaloklossia formed a sister group to the Sarcocystis spp. Sarcocystis species were divided into three main lineages; S. neurona isolates were located in the second lineage and clustered with S. mucosa, S. dispersa, S. lacertae, S. rodentifelis, S. muris, and Frenkelia spp. Alignment of S. neurona SSU rRNA gene sequences of Michigan opossum isolates (MIOP5, MIOP20) and a S. neurona Michigan horse isolate (MIH8) showed 100% identity. These Michigan isolates differed in 2/1085 bp (0.2%) from a Kentucky S. neurona horse isolate (SN5). Additionally, S. neurona isolates from horses and opossums were identical based on the ultrastructural features and PCR-RFLP analyses thus forming a phylogenetically indistinct group in these regions. These findings revealed the concordance between the morphological and molecular data and confirmed that S. neurona from opossums and horses originated from the same phylogenetic origin.

  1. Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

    NASA Astrophysics Data System (ADS)

    Decho, Alan W.; Beckman, Erin M.; Chandler, G. Thomas; Kawaguchi, Tomohiro

    2008-06-01

    An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.

  2. Office space bacterial abundance and diversity in three metropolitan areas.

    PubMed

    Hewitt, Krissi M; Gerba, Charles P; Maxwell, Sheri L; Kelley, Scott T

    2012-01-01

    People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded "universal" bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. "[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay." - Feazel et al. (2009).

  3. Bacterial Degraders of Coexisting Dichloromethane, Benzene, and Toluene, Identified by Stable-Isotope Probing.

    PubMed

    Yoshikawa, Miho; Zhang, Ming; Kurisu, Futoshi; Toyota, Koki

    2017-01-01

    Most bioremediation studies on volatile organic compounds (VOCs) have focused on a single contaminant or its derived compounds and degraders have been identified under single contaminant conditions. Bioremediation of multiple contaminants remains a challenging issue. To identify a bacterial consortium that degrades multiple VOCs (dichloromethane (DCM), benzene, and toluene), we applied DNA-stable isotope probing. For individual tests, we combined a 13 C-labeled VOC with other two unlabeled VOCs, and prepared three unlabeled VOCs as a reference. Over 11 days, DNA was periodically extracted from the consortia, and the bacterial community was evaluated by next-generation sequencing of bacterial 16S rRNA gene amplicons. Density gradient fractions of the DNA extracts were amplified by universal bacterial primers for the 16S rRNA gene sequences, and the amplicons were analyzed by terminal restriction fragment length polymorphism (T-RFLP) using restriction enzymes: Hha I and Msp I. The T-RFLP fragments were identified by 16S rRNA gene cloning and sequencing. Under all test conditions, the consortia were dominated by Rhodanobacter , Bradyrhizobium / Afipia , Rhizobium , and Hyphomicrobium . DNA derived from Hyphomicrobium and Propioniferax shifted toward heavier fractions under the condition added with 13 C-DCM and 13 C-benzene, respectively, compared with the reference, but no shifts were induced by 13 C-toluene addition. This implies that Hyphomicrobium and Propioniferax were the main DCM and benzene degraders, respectively, under the coexisting condition. The known benzene degrader Pseudomonas sp. was present but not actively involved in the degradation.

  4. Characterizing the bacterial microbiota in different gastrointestinal tract segments of the Bactrian camel.

    PubMed

    He, Jing; Yi, Li; Hai, Le; Ming, Liang; Gao, Wanting; Ji, Rimutu

    2018-01-12

    The bacterial community plays important roles in the gastrointestinal tracts (GITs) of animals. However, our understanding of the microbial communities in the GIT of Bactrian camels remains limited. Here, we describe the bacterial communities from eight different GIT segments (rumen, reticulum, abomasum, duodenum, ileum, jejunum, caecum, colon) and faeces determined from 11 Bactrian camels using 16S rRNA gene amplicon sequencing. Twenty-seven bacterial phyla were found in the GIT, with Firmicutes, Verrucomicrobia and Bacteroidetes predominating. However, there were significant differences in microbial community composition between segments of the GIT. In particular, a greater proportion of Akkermansia and Unclassified Ruminococcaceae were found in the large intestine and faecal samples, while more Unclassified Clostridiales and Unclassified Bacteroidales were present in the in forestomach and small intestine. Comparative analysis of the microbiota from different GIT segments revealed that the microbial profile in the large intestine was like that in faeces. We also predicted the metagenomic profiles for the different GIT regions. In forestomach, there was enrichment associated with replication and repair and amino acid metabolism, while carbohydrate metabolism was enriched in the large intestine and faeces. These results provide profound insights into the GIT microbiota of Bactrian camels.

  5. Diversity of Bacterial Communities in Container Habitats of Mosquitoes

    PubMed Central

    Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

    2010-01-01

    We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

  6. Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

    PubMed

    Ceuppens, Siele; De Coninck, Dieter; Bottledoorn, Nadine; Van Nieuwerburgh, Filip; Uyttendaele, Mieke

    2017-09-18

    Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results. Copyright © 2017 Elsevier B

  7. Methane production and small intestinal bacterial overgrowth in children living in a slum.

    PubMed

    Mello, Carolina Santos; Tahan, Soraia; Melli, Lígia Cristina F L; Rodrigues, Mirian Silva do Carmo; de Mello, Ricardo Martin Pereira; Scaletsky, Isabel Cristina Affonso; de Morais, Mauro Batista

    2012-11-07

    To analyze small intestinal bacterial overgrowth in school-aged children and the relationship between hydrogen and methane production in breath tests. This transversal study included 85 children residing in a slum and 43 children from a private school, all aged between 6 and 10 years, in Osasco, Brazil. For characterization of the groups, data regarding the socioeconomic status and basic housing sanitary conditions were collected. Anthropometric data was obtained in children from both groups. All children completed the hydrogen (H(2)) and methane (CH(4)) breath test in order to assess small intestinal bacterial overgrowth (SIBO). SIBO was diagnosed when there was an increase in H(2) ≥ 20 ppm or CH(4) ≥ 10 ppm with regard to the fasting value until 60 min after lactulose ingestion. Children from the slum group had worse living conditions and lower nutritional indices than children from the private school. SIBO was found in 30.9% (26/84) of the children from the slum group and in 2.4% (1/41) from the private school group (P = 0.0007). Greater hydrogen production in the small intestine was observed in children from the slum group when compared to children from the private school (P = 0.007). A higher concentration of hydrogen in the small intestine (P < 0.001) and in the colon (P < 0.001) was observed among the children from the slum group with SIBO when compared to children from the slum group without SIBO. Methane production was observed in 63.1% (53/84) of the children from the slum group and in 19.5% (8/41) of the children from the private school group (P < 0.0001). Methane production was observed in 38/58 (65.5%) of the children without SIBO and in 15/26 (57.7%) of the children with SIBO from the slum. Colonic production of hydrogen was lower in methane-producing children (P = 0.017). Children who live in inadequate environmental conditions are at risk of bacterial overgrowth and methane production. Hydrogen is a substrate for methane production in the colon.

  8. Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

    PubMed

    Teixeira, M M; Campaner, M; Camargo, E P

    1994-01-01

    To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

  9. El Verde Ridge, El Verde Valley, and Rio Icacos root phosphatase and bacterial community composition (December 2015)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cabugao, Kristine; Timm, Collin; Carrell, Alyssa

    Raw data of resin P values, root phosphatase, bacterial community 16S rRNA gene sequences, and bacterial isolate phosphatase and P solubilization in Rio Icacos, El Verde Ridge and El Verde Valley. Contact cabugaokm@ornl.gov if you need to use this dataset for additional information.

  10. AtPAP2 modulates the import of the small subunit of Rubisco into chloroplasts.

    PubMed

    Zhang, Renshan; Guan, Xiaoqian; Law, Yee-Song; Sun, Feng; Chen, Shuai; Wong, Kam Bo; Lim, Boon Leong

    2016-10-02

    Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.

  11. Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

    PubMed Central

    2015-01-01

    The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration. PMID:24661308

  12. Subunit interface dynamics in hexadecameric rubisco.

    PubMed

    van Lun, Michiel; van der Spoel, David; Andersson, Inger

    2011-09-02

    Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

    PubMed

    McGarvey, J A; Franco, R B; Palumbo, J D; Hnasko, R; Stanker, L; Mitloehner, F M

    2013-06-01

    To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes. © 2013 The Society for Applied Microbiology No claim to US Government works.

  14. GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis.

    PubMed

    Wang, Si-Qi; Shi, Dong-Qiao; Long, Yan-Ping; Liu, Jie; Yang, Wei-Cai

    2012-01-01

    RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.

  15. Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.

    PubMed

    Yokoyama, Keiko; Doi, Yohei; Yamane, Kunikazu; Kurokawa, Hiroshi; Shibata, Naohiro; Shibayama, Keigo; Yagi, Tetsuya; Kato, Haru; Arakawa, Yoshichika

    2003-12-06

    Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

  16. The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions.

    PubMed

    Mio, Kazuhiro; Mio, Muneyo; Arisaka, Fumio; Sato, Masahiko; Sato, Chikara

    2010-09-01

    The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation. 2010 Elsevier Ltd. All rights reserved.

  17. Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change

    PubMed Central

    Kaartokallio, Hermanni; Laamanen, Maria; Sivonen, Kaarina

    2005-01-01

    To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0°C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the α- and γ-proteobacteria or the Cytophaga-Flavobacterium-Bacteroides group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure. PMID:16085826

  18. Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

    PubMed Central

    Preveena, Jagadesan; Bhore, Subhash J.

    2013-01-01

    Background: In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. Objective: The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. Materials and Methods: Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. Results: Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. Conclusion: Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study. PMID:24501447

  19. Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

    PubMed

    Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

    2008-04-01

    To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

  20. Culture-Independent Analysis of Aerosol Microbiology in a Metropolitan Subway System

    PubMed Central

    Robertson, Charles E.; Baumgartner, Laura K.; Harris, J. Kirk; Peterson, Kristen L.; Stevens, Mark J.; Frank, Daniel N.

    2013-01-01

    The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ∼75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events. PMID:23542619

  1. Culture-independent analysis of aerosol microbiology in a metropolitan subway system.

    PubMed

    Robertson, Charles E; Baumgartner, Laura K; Harris, J Kirk; Peterson, Kristen L; Stevens, Mark J; Frank, Daniel N; Pace, Norman R

    2013-06-01

    The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ~75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events.

  2. Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem.

    PubMed

    Beyter, Doruk; Tang, Pei-Zhong; Becker, Scott; Hoang, Tony; Bilgin, Damla; Lim, Yan Wei; Peterson, Todd C; Mayfield, Stephen; Haerizadeh, Farzad; Shurin, Jonathan B; Bafna, Vineet; McBride, Robert

    2016-04-01

    Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

    PubMed Central

    Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

    2002-01-01

    Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

  4. Stability of a biogas-producing bacterial, archaeal and fungal community degrading food residues.

    PubMed

    Bengelsdorf, Frank R; Gerischer, Ulrike; Langer, Susanne; Zak, Manuel; Kazda, Marian

    2013-04-01

    The resident microbiota was analyzed in a mesophilic, continuously operating biogas plant predominantly utilizing food residues, stale bread, and other waste cosubstrates together with pig manure and maize silage. The dominating bacterial, archaeal, and eukaryotic community members were characterized by two different 16S/18S rRNA gene culture-independent approaches. Prokaryotic 16S rRNA gene and eukaryotic 18S rRNA gene clone libraries were constructed and further analyzed by restriction fragment length polymorphism (RFLP), 16S/18S rRNA gene sequencing, and phylogenetic tree reconstruction. The most dominant bacteria belonged to the phyla Bacteriodetes, Chloroflexus, and Firmicutes. On the family level, the bacterial composition confirmed high differences among biogas plants studied so fare. In contrast, the methanogenic archaeal community was similar to that of other studied biogas plants. Furthermore, it was possible to identify fungi at the genus level, namely Saccharomyces and Mucor. Both genera, which are important for microbial degradation of complex compounds, were up to now not found in biogas plants. The results revealed their long-term presence as indicated by denaturating gradient gel electrophoresis (DGGE). The DGGE method confirmed that the main members of the microbial community were constantly present over more than one-year period. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. Changes in Soil Bacterial Communities and Diversity in ...

    EPA Pesticide Factsheets

    Silver-induced selective pressure is becoming increasingly important due to the growing use of silver (Ag) as an antimicrobial agent in biomedical and commercial products. With demonstrated links between environmental resistomes and clinical pathogens, it is important to identify microbial profiles related to silver tolerance/resistance. We investigated the effects of ionic Ag stress on soil bacterial communities and identified resistant/persistant bacterial populations. Silver treatments of 50 - 400 mg Ag kg-1 soil were established in five soils. Chemical lability measurements using diffusive gradients in thin-film devices confirmed that significant (albeit decreasing) labile Ag concentrations were present throughout the 9-month incubation period. Synchrotron X-ray absorption near edge structure spectroscopy demonstrate that this decreasing lability was due to changes in Ag speciation to less soluble forms such as Ag0 and Ag2S. Real-time PCR and Illumina MiSeq screening of 16S rRNA bacterial genes showed β-diversity in response to Ag pressure, and immediate and significant reductions in 16S rRNA gene counts with varying degrees of recovery. These effects were more strongly influenced by exposure time than by Ag dose at these rates. Ag-selected dominant OTUs principally resided in known persister taxa (mainly Gram positive), including metal-tolerant bacteria and slow-growing Mycobacteria. Soil microbial communities have been implicated as sources of an

  6. Cutaneous manifestations of Crohn's disease, its spectrum, and its pathogenesis: intracellular consensus bacterial 16S rRNA is associated with the gastrointestinal but not the cutaneous manifestations of Crohn's disease.

    PubMed

    Crowson, A Neil; Nuovo, Gerard J; Mihm, Martin C; Magro, Cynthia

    2003-11-01

    The classic pathology of skin disease discontinuous from the inflamed gastrointestinal (GI) tract in patients with Crohn's disease (CD) includes pyoderma gangrenosum (PG), erythema nodosum (EN), and so-called metastatic Crohn's disease. The purpose of this study was two-fold: First, we explored the full spectrum of cutaneous lesions associated with Crohn's disease, and second, we sought to explore a potential molecular basis of the skin lesions in patients with CD. In this regard, we analyzed skin and GI tract biopsies from affected patients for the consensus bacterial SrRNA to determine whether direct bacterial infection was associated with either condition. Formalin-fixed, paraffin-embedded sections were studied and correlated to clinical presentation and histories from 33 patients with CD. Consensus bacterial RNA sequences were analyzed using an RT in situ PCR assay on both skin biopsy and GI biopsy material. The GI tract material included biopsies from 3 patients who had skin lesions and from 7 patients in whom there were no known skin manifestations. There were 8 cases of neutrophilic dominant dermal infiltrates, including pyoderma gangrenosum, 6 cases of granuloma annulare/necrobiosis lipoidica-like lesions, 5 cases of sterile neutrophilic folliculitis, 5 cases of panniculitis, 4 cases of vasculitis, 2 cases of psoriasis, 2 cases of lichenoid and granulomatous inflammation, and 1 case of classic metastatic CD. Intracellular bacterial 16S rRNA was detected in 8 of 10 tissues of active CD in the GI tract, of which 3 of the cases tested were from patients who also developed skin lesions at some point in their clinical course; in contrast, none of the skin biopsies had detectable bacterial RNA. The dermatopathological manifestations of CD discontiguous from the involved GI tract mucosa have in common a vascular injury syndrome, typically with a prominent extravascular neutrophilic and/or histiocytic dermal infiltrate. In addition, this study, the first to

  7. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

    PubMed

    Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

    2000-04-07

    Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into

  8. Linking soil bacterial biodiversity and soil carbon stability.

    PubMed

    Mau, Rebecca L; Liu, Cindy M; Aziz, Maliha; Schwartz, Egbert; Dijkstra, Paul; Marks, Jane C; Price, Lance B; Keim, Paul; Hungate, Bruce A

    2015-06-01

    Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this 'priming' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability.

  9. Bacterial microbiome and nematode occurrence in different potato agricultural soils

    USDA-ARS?s Scientific Manuscript database

    Pratylenchus neglectus and Meloidogyne chitwoodi are the main plant-parasitic nematodes in potato crops of the San Luis Valley, Colorado. Bacterial microbiome (16S rRNA copies per gram of soil) and nematode communities (nematodes per 200 gr of soil) from five different potato farms were analyzed to ...

  10. Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms

    PubMed Central

    Hochrein, Hubertus; Kirschning, Carsten J.

    2013-01-01

    The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13. PMID:23802068

  11. Prediction of bacterial small RNAs in the RsmA (CsrA) and ToxT pathways: a machine learning approach.

    PubMed

    Fakhry, Carl Tony; Kulkarni, Prajna; Chen, Ping; Kulkarni, Rahul; Zarringhalam, Kourosh

    2017-08-22

    Small RNAs (sRNAs) constitute an important class of post-transcriptional regulators that control critical cellular processes in bacteria. Recent research using high-throughput transcriptomic approaches has led to a dramatic increase in the discovery of bacterial sRNAs. However, it is generally believed that the currently identified sRNAs constitute a limited subset of the bacterial sRNA repertoire. In several cases, sRNAs belonging to a specific class are already known and the challenge is to identify additional sRNAs belonging to the same class. In such cases, machine-learning approaches can be used to predict novel sRNAs in a given class. In this work, we develop novel bioinformatics approaches that integrate sequence and structure-based features to train machine-learning models for the discovery of bacterial sRNAs. We show that features derived from recurrent structural motifs in the ensemble of low energy secondary structures can distinguish the RNA classes with high accuracy. We apply this approach to predict new members in two broad classes of bacterial small RNAs: 1) sRNAs that bind to the RNA-binding protein RsmA/CsrA in diverse bacterial species and 2) sRNAs regulated by the master regulator of virulence, ToxT, in Vibrio cholerae. The involvement of sRNAs in bacterial adaptation to changing environments is an increasingly recurring theme in current research in microbiology. It is likely that future research, combining experimental and computational approaches, will discover many more examples of sRNAs as components of critical regulatory pathways in bacteria. We have developed a novel approach for prediction of small RNA regulators in important bacterial pathways. This approach can be applied to specific classes of sRNAs for which several members have been identified and the challenge is to identify additional sRNAs.

  12. Variability of Bacterial Communities in the Moth Heliothis virescens Indicates Transient Association with the Host

    PubMed Central

    Staudacher, Heike; Kaltenpoth, Martin; Breeuwer, Johannes A. J.; Menken, Steph B. J.; Heckel, David G.; Groot, Astrid T.

    2016-01-01

    Microbes associated with insects can confer a wide range of ecologically relevant benefits to their hosts. Since insect-associated bacteria often increase the nutritive value of their hosts' diets, the study of bacterial communities is especially interesting in species that are important agricultural pests. We investigated the composition of bacterial communities in the noctuid moth Heliothis virescens and its variability in relation to developmental stage, diet and population (field and laboratory), using bacterial tag-encoded FLX pyrosequencing of 16S rRNA amplicons. In larvae, bacterial communities differed depending on the food plant on which they had been reared, although the within-group variation between biological replicates was high as well. Moreover, larvae originating from a field or laboratory population did not share any OTUs. Interestingly, Enterococcus sp. was found to be the dominant taxon in laboratory-reared larvae, but was completely absent from field larvae, indicating dramatic shifts in microbial community profiles upon cultivation of the moths in the laboratory. Furthermore, microbiota composition varied strongly across developmental stages in individuals of the field population, and we found no evidence for vertical transmission of bacteria from mothers to offspring. Since sample sizes in our study were small due to pooling of samples for sequencing, we cautiously conclude that the high variability in bacterial communities suggests a loose and temporary association of the identified bacteria with H. virescens. PMID:27139886

  13. Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity.

    PubMed

    Balcázar, José L; Lee, Natuschka M; Pintado, José; Planas, Miquel

    2010-03-01

    Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described. Crown Copyright 2010. Published by Elsevier GmbH. All rights reserved.

  14. Bacterial community composition characterization of a lead-contaminated Microcoleus sp. consortium.

    PubMed

    Giloteaux, Ludovic; Solé, Antoni; Esteve, Isabel; Duran, Robert

    2011-08-01

    A Microcoleus sp. consortium, obtained from the Ebro delta microbial mat, was maintained under different conditions including uncontaminated, lead-contaminated, and acidic conditions. Terminal restriction fragment length polymorphism and 16S rRNA gene library analyses were performed in order to determine the effect of lead and culture conditions on the Microcoleus sp. consortium. The bacterial composition inside the consortium revealed low diversity and the presence of specific terminal-restriction fragments under lead conditions. 16S rRNA gene library analyses showed that members of the consortium were affiliated to the Alpha, Beta, and Gammaproteobacteria and Cyanobacteria. Sequences closely related to Achromobacter spp., Alcaligenes faecalis, and Thiobacillus species were exclusively found under lead conditions while sequences related to Geitlerinema sp., a cyanobacterium belonging to the Oscillatoriales, were not found in presence of lead. This result showed a strong lead selection of the bacterial members present in the Microcoleus sp. consortium. Several of the 16S rRNA sequences were affiliated to nitrogen-fixing microorganisms including members of the Rhizobiaceae and the Sphingomonadaceae. Additionally, confocal laser scanning microscopy and scanning and transmission electron microscopy showed that under lead-contaminated condition Microcoleus sp. cells were grouped and the number of electrodense intracytoplasmic inclusions was increased.

  15. Microbiome analysis reveals the abundance of bacterial pathogens in Rousettus leschenaultii guano

    PubMed Central

    Banskar, Sunil; Bhute, Shrikant S.; Suryavanshi, Mangesh V.; Punekar, Sachin; Shouche, Yogesh S.

    2016-01-01

    Bats are crucial for proper functioning of an ecosystem. They provide various important services to ecosystem and environment. While, bats are well-known carrier of pathogenic viruses, their possible role as a potential carrier of pathogenic bacteria is under-explored. Here, using culture-based approach, employing multiple bacteriological media, over thousand bacteria were cultivated and identified from Rousettus leschenaultii (a frugivorous bat species), the majority of which were from the family Enterobacteriaceae and putative pathogens. Next, pathogenic potential of most frequently cultivated component of microbiome i.e. Escherichia coli was assessed to identify its known pathotypes which revealed the presence of virulent factors in many cultivated E. coli isolates. Applying in-depth bacterial community analysis using high-throughput 16 S rRNA gene sequencing, a high inter-individual variation was observed among the studied guano samples. Interestingly, a higher diversity of bacterial communities was observed in decaying guano representative. The search against human pathogenic bacteria database at 97% identity, a small proportion of sequences were found associated to well-known human pathogens. The present study thus indicates that this bat species may carry potential bacterial pathogens and advice to study the effect of these pathogens on bats itself and the probable mode of transmission to humans and other animals. PMID:27845426

  16. Microbiome analysis reveals the abundance of bacterial pathogens in Rousettus leschenaultii guano.

    PubMed

    Banskar, Sunil; Bhute, Shrikant S; Suryavanshi, Mangesh V; Punekar, Sachin; Shouche, Yogesh S

    2016-11-15

    Bats are crucial for proper functioning of an ecosystem. They provide various important services to ecosystem and environment. While, bats are well-known carrier of pathogenic viruses, their possible role as a potential carrier of pathogenic bacteria is under-explored. Here, using culture-based approach, employing multiple bacteriological media, over thousand bacteria were cultivated and identified from Rousettus leschenaultii (a frugivorous bat species), the majority of which were from the family Enterobacteriaceae and putative pathogens. Next, pathogenic potential of most frequently cultivated component of microbiome i.e. Escherichia coli was assessed to identify its known pathotypes which revealed the presence of virulent factors in many cultivated E. coli isolates. Applying in-depth bacterial community analysis using high-throughput 16 S rRNA gene sequencing, a high inter-individual variation was observed among the studied guano samples. Interestingly, a higher diversity of bacterial communities was observed in decaying guano representative. The search against human pathogenic bacteria database at 97% identity, a small proportion of sequences were found associated to well-known human pathogens. The present study thus indicates that this bat species may carry potential bacterial pathogens and advice to study the effect of these pathogens on bats itself and the probable mode of transmission to humans and other animals.

  17. Use of 16S rRNA Sequencing for Identification of Actinobacillus ureae Isolated from a Cerebrospinal Fluid Sample

    PubMed Central

    Whitelaw, A. C.; Shankland, I. M.; Elisha, B. G.

    2002-01-01

    Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene. PMID:11825992

  18. Biphasic Study to Characterize Agricultural Biogas Plants by High-Throughput 16S rRNA Gene Amplicon Sequencing and Microscopic Analysis.

    PubMed

    Maus, Irena; Kim, Yong Sung; Wibberg, Daniel; Stolze, Yvonne; Off, Sandra; Antonczyk, Sebastian; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

    2017-02-28

    Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus , were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

  19. Bacterial microbiome of the nose of healthy dogs and dogs with nasal disease

    PubMed Central

    Dorn, Elisabeth S.; Suchodolski, Jan S.; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S.

    2017-01-01

    The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease. PMID:28459886

  20. Bacterial microbiome of the nose of healthy dogs and dogs with nasal disease.

    PubMed

    Tress, Barbara; Dorn, Elisabeth S; Suchodolski, Jan S; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S

    2017-01-01

    The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease.

  1. Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

    PubMed Central

    Uhlemann, Eva-Maria E; Pierson, Hannah E; Fillingame, Robert H; Dmitriev, Oleg Y

    2012-01-01

    NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes. PMID:22162071

  2. Microbial Diversity in Commercial Bee Pollen from Europe, Chile, and Mexico, Based on 16S rRNA Gene Amplicon Metagenome Sequencing

    PubMed Central

    Moreno Andrade, Vicente D.; Saldaña Gutiérrez, Carlos; Calvillo Medina, Rosa P.; Cruz Hérnandez, Andrés; Vázquez Cruz, Moisés A.; Torres Ruíz, Alfonso; Romero Gómez, Sergio; Ramos López, Miguel A.; Álvarez-Hidalgo, Erika; López-Gaytan, Silvia B.; Ramírez, Natanahel Salvador; Jones, George H.

    2018-01-01

    ABSTRACT Bee pollen is a highly nutritive natural foodstuff. Because of its use as a comestible, the association of bacteria with bee pollen is commercially and biologically important. We report here the bacterial diversity of seven bee pollen samples (five from Europe, one from Chile, and one from Mexico) based on 16S rRNA gene amplicon metagenome sequencing. PMID:29773615

  3. RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.

    PubMed

    Lindahl, Lasse; Bommankanti, Ananth; Li, Xing; Hayden, Lauren; Jones, Adrienne; Khan, Miriam; Oni, Tolulope; Zengel, Janice M

    2009-07-01

    RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

  4. Genetic diversity among Babesia rossi detected in naturally infected dogs in Abeokuta, Nigeria, based on 18S rRNA gene sequences.

    PubMed

    Takeet, Michael I; Oyewusi, Adeoye J; Abakpa, Simon A V; Daramola, Olukayode O; Peters, Sunday O

    2017-03-01

    Adequate knowledge of the genetic diversity among Babesia species infecting dogs is necessary for a better understanding of the epidemiology and control of canine babesiosis. Hence, this study determined the genetic diversity among the Babesia rossi detected in dogs presented for routine examination in Veterinary Hospitals in Abeokuta, Nigeria. Blood were randomly collected from 209 dogs. Field-stained thin smears were made and DNA extracted from the blood. Partial region of the 18S small subunit ribosomal RNA (rRNA) gene was amplified, sequenced and analysed. Babesia species was detected in 16 (7.7%) of the dogs by microscopy. Electrophoresed PCR products from 39 (18.66%) dogs revealed band size of 450 bp and 2 (0.95%) dogs had band size of 430 bp. The sequences obtained from 450 bp amplicon displayed homology of 99.74% (387/388) with partial sequences of 18S rRNA gene of Babesia rossi in the GeneBank. Of the two sequences that had 430 bp amplicon, one was identified as T. annulata and second as T. ovis. A significantly (p<0.05) higher prevalence of B. rossi was detected by PCR compared to microscopy. The mean PCV of Babesia infected dogs was significantly (p<0.05) lower than non-infected dogs. Phylogenetic analysis revealed minimal diversity among B. rossi with the exception of one sequence that was greatly divergent from the others. This study suggests that more than one genotype of B. rossi may be in circulation among the dog population in the study area and this may have potential implication on clinical outcome of canine babesiosis.

  5. New rRNA Gene-Based Phylogenies of the Alphaproteobacteria Provide Perspective on Major Groups, Mitochondrial Ancestry and Phylogenetic Instability

    PubMed Central

    Ferla, Matteo P.; Thrash, J. Cameron; Giovannoni, Stephen J.; Patrick, Wayne M.

    2013-01-01

    Bacteria in the class Alphaproteobacteria have a wide variety of lifestyles and physiologies. They include pathogens of humans and livestock, agriculturally valuable strains, and several highly abundant marine groups. The ancestor of mitochondria also originated in this clade. Despite significant effort to investigate the phylogeny of the Alphaproteobacteria with a variety of methods, there remains considerable disparity in the placement of several groups. Recent emphasis on phylogenies derived from multiple protein-coding genes remains contentious due to disagreement over appropriate gene selection and the potential influences of systematic error. We revisited previous investigations in this area using concatenated alignments of the small and large subunit (SSU and LSU) rRNA genes, as we show here that these loci have much lower GC bias than whole genomes. This approach has allowed us to update the canonical 16S rRNA gene tree of the Alphaproteobacteria with additional important taxa that were not previously included, and with added resolution provided by concatenating the SSU and LSU genes. We investigated the topological stability of the Alphaproteobacteria by varying alignment methods, rate models, taxon selection and RY-recoding to circumvent GC content bias. We also introduce RYMK-recoding and show that it avoids some of the information loss in RY-recoding. We demonstrate that the topology of the Alphaproteobacteria is sensitive to inclusion of several groups of taxa, but it is less affected by the choice of alignment and rate methods. The majority of topologies and comparative results from Approximately Unbiased tests provide support for positioning the Rickettsiales and the mitochondrial branch within a clade. This composite clade is a sister group to the abundant marine SAR11 clade (Pelagibacterales). Furthermore, we add support for taxonomic assignment of several recently sequenced taxa. Accordingly, we propose three subclasses within the

  6. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The

  7. Methane production and small intestinal bacterial overgrowth in children living in a slum

    PubMed Central

    Mello, Carolina Santos; Tahan, Soraia; Melli, Lígia Cristina FL; Rodrigues, Mirian Silva do Carmo; de Mello, Ricardo Martin Pereira; Scaletsky, Isabel Cristina Affonso; de Morais, Mauro Batista

    2012-01-01

    AIM: To analyze small intestinal bacterial overgrowth in school-aged children and the relationship between hydrogen and methane production in breath tests. METHODS: This transversal study included 85 children residing in a slum and 43 children from a private school, all aged between 6 and 10 years, in Osasco, Brazil. For characterization of the groups, data regarding the socioeconomic status and basic housing sanitary conditions were collected. Anthropometric data was obtained in children from both groups. All children completed the hydrogen (H2) and methane (CH4) breath test in order to assess small intestinal bacterial overgrowth (SIBO). SIBO was diagnosed when there was an increase in H2 ≥ 20 ppm or CH4 ≥ 10 ppm with regard to the fasting value until 60 min after lactulose ingestion. RESULTS: Children from the slum group had worse living conditions and lower nutritional indices than children from the private school. SIBO was found in 30.9% (26/84) of the children from the slum group and in 2.4% (1/41) from the private school group (P = 0.0007). Greater hydrogen production in the small intestine was observed in children from the slum group when compared to children from the private school (P = 0.007). A higher concentration of hydrogen in the small intestine (P < 0.001) and in the colon (P < 0.001) was observed among the children from the slum group with SIBO when compared to children from the slum group without SIBO. Methane production was observed in 63.1% (53/84) of the children from the slum group and in 19.5% (8/41) of the children from the private school group (P < 0.0001). Methane production was observed in 38/58 (65.5%) of the children without SIBO and in 15/26 (57.7%) of the children with SIBO from the slum. Colonic production of hydrogen was lower in methane-producing children (P = 0.017). CONCLUSION: Children who live in inadequate environmental conditions are at risk of bacterial overgrowth and methane production. Hydrogen is a substrate for

  8. Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae, Prorocentrales).

    PubMed

    Boopathi, Thangavelu; Faria, Daphne Georgina; Cheon, Ju-Yong; Youn, Seok Hyun; Ki, Jang-Seu

    2015-01-01

    The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one-factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t-test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  9. Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution.

    PubMed

    Yonemoto, Isaac T; Matteri, Christopher W; Nguyen, Thao Amy; Smith, Hamilton O; Weyman, Philip D

    2013-07-02

    Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [Ni

  10. Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

    PubMed Central

    2013-01-01

    Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe

  11. The Role of Chaperone-subunit Usher Domain Interactions in the Mechanism of Bacterial Pilus Biogenesis Revealed by ESI-MS*

    PubMed Central

    Morrissey, Bethny; Leney, Aneika C.; Toste Rêgo, Ana; Phan, Gilles; Allen, William J.; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E.; Radford, Sheena E.

    2012-01-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species. PMID:22371487

  12. The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.

    PubMed

    Morrissey, Bethny; Leney, Aneika C; Toste Rêgo, Ana; Phan, Gilles; Allen, William J; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E; Radford, Sheena E

    2012-07-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.

  13. Bacterial Community Dynamics in Dichloromethane-Contaminated Groundwater Undergoing Natural Attenuation

    PubMed Central

    Wright, Justin; Kirchner, Veronica; Bernard, William; Ulrich, Nikea; McLimans, Christopher; Campa, Maria F.; Hazen, Terry; Macbeth, Tamzen; Marabello, David; McDermott, Jacob; Mackelprang, Rachel; Roth, Kimberly; Lamendella, Regina

    2017-01-01

    The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. This study investigates the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 μg/L. Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. Across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges

  14. Bacterial Community Dynamics in Dichloromethane-Contaminated Groundwater Undergoing Natural Attenuation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wright, Justin; Kirchner, Veronica; Bernard, William

    The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. Here, we investigate the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 μg/L.more » Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. And across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges

  15. Bacterial Community Dynamics in Dichloromethane-Contaminated Groundwater Undergoing Natural Attenuation

    DOE PAGES

    Wright, Justin; Kirchner, Veronica; Bernard, William; ...

    2017-11-22

    The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. Here, we investigate the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 μg/L.more » Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. And across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges

  16. Initial insights into bacterial succession during human decomposition.

    PubMed

    Hyde, Embriette R; Haarmann, Daniel P; Petrosino, Joseph F; Lynne, Aaron M; Bucheli, Sibyl R

    2015-05-01

    Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession.

  17. Developing an Apicomplexan DNA Barcoding System to Detect Blood Parasites of Small Coral Reef Fishes.

    PubMed

    Renoux, Lance P; Dolan, Maureen C; Cook, Courtney A; Smit, Nico J; Sikkel, Paul C

    2017-08-01

    Apicomplexan parasites are obligate parasites of many species of vertebrates. To date, there is very limited understanding of these parasites in the most-diverse group of vertebrates, actinopterygian fishes. While DNA barcoding targeting the eukaryotic 18S small subunit rRNA gene sequence has been useful in identifying apicomplexans in tetrapods, identification of apicomplexans infecting fishes has relied solely on morphological identification by microscopy. In this study, a DNA barcoding method was developed that targets the 18S rRNA gene primers for identifying apicomplexans parasitizing certain actinopterygian fishes. A lead primer set was selected showing no cross-reactivity to the overwhelming abundant host DNA and successfully confirmed 37 of the 41 (90.2%) microscopically verified parasitized fish blood samples analyzed in this study. Furthermore, this DNA barcoding method identified 4 additional samples that screened negative for parasitemia, suggesting this molecular method may provide improved sensitivity over morphological characterization by microscopy. In addition, this PCR screening method for fish apicomplexans, using Whatman FTA preserved DNA, was tested in efforts leading to a more simplified field collection, transport, and sample storage method as well as a streamlining sample processing important for DNA barcoding of large sample sets.

  18. The complete mitochondrial genome sequence of Eimeria innocua (Eimeriidae, Coccidia, Apicomplexa).

    PubMed

    Hafeez, Mian Abdul; Vrba, Vladimir; Barta, John Robert

    2016-07-01

    The complete mitochondrial genome of Eimeria innocua KR strain (Eimeriidae, Coccidia, Apicomplexa) was sequenced. This coccidium infects turkeys (Meleagris gallopavo), Bobwhite quails (Colinus virginianus), and Grey partridges (Perdix perdix). Genome organization and gene contents were comparable with other Eimeria spp. infecting galliform birds. The circular-mapping mt genome of E. innocua is 6247 bp in length with three protein-coding genes (cox1, cox3, and cytb), 19 gene fragments encoding large subunit (LSU) rRNA and 14 gene fragments encoding small subunit (SSU) rRNA. Like other Apicomplexa, no tRNA was encoded. The mitochondrial genome of E. innocua confirms its close phylogenetic affinities to Eimeria dispersa.

  19. Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification.

    PubMed

    Yao, Yongpeng; Li, Shanshan; Cao, Jiaqian; Liu, Weiwei; Fan, Keqiang; Xiang, Wensheng; Yang, Keqian; Kong, Deming; Wang, Weishan

    2018-05-08

    Here, we demonstrate an easy-to-implement and general biosensing strategy by coupling the small-molecule recognition of the bacterial allosteric transcription factor (aTF) with isothermal strand displacement amplification (SDA) in vitro. Based on this strategy, we developed two biosensors for the detection of an antiseptic, p-hydroxybenzoic acid, and a disease marker, uric acid, using bacterial aTF HosA and HucR, respectively, highlighting the great potential of this strategy for the development of small-molecule biosensors.

  20. Unusual bacterioplankton community structure in ultra-oligotrophic Crater Lake

    USGS Publications Warehouse

    Urbach, Ena; Vergin, Kevin L.; Morse, Ariel

    2001-01-01

    The bacterioplankton assemblage in Crater Lake, Oregon (U.S.A.), is different from communities found in other oxygenated lakes, as demonstrated by four small subunit ribosomal ribonucleic acid (SSU rRNA) gene clone libraries and oligonucleotide probe hybridization to RNA from lake water. Populations in the euphotic zone of this deep (589 m), oligotrophic caldera lake are dominated by two phylogenetic clusters of currently uncultivated bacteria: CL120-10, a newly identified cluster in the verrucomicrobiales, and ACK4 actinomycetes, known as a minor constituent of bacterioplankton in other lakes. Deep-water populations at 300 and 500 m are dominated by a different pair of uncultivated taxa: CL500-11, a novel cluster in the green nonsulfur bacteria, and group I marine crenarchaeota. b-Proteobacteria, dominant in most other freshwater environments, are relatively rare in Crater Lake (<=16% of nonchloroplast bacterial rRNA at all depths). Other taxa identified in Crater Lake libraries include a newly identified candidate bacterial division, ABY1, and a newly identified subcluster, CL0-1, within candidate division OP10. Probe analyses confirmed vertical stratification of several microbial groups, similar to patterns observed in open-ocean systems. Additional similarities between Crater Lake and ocean microbial populations include aphotic zone dominance of group I marine crenarchaeota and green nonsulfur bacteria. Comparison of Crater Lake to other lakes studied by rRNA methods suggests that selective factors structuring Crater Lake bacterioplankton populations may include low concentrations of available trace metals and dissolved organic matter, chemistry of infiltrating hydrothermal waters, and irradiation by high levels of ultraviolet light.

  1. Soil-Borne Bacterial Structure and Diversity Does Not Reflect Community Activity in Pampa Biome

    PubMed Central

    Lupatini, Manoeli; Suleiman, Afnan Khalil Ahmad; Jacques, Rodrigo Josemar Seminoti; Antoniolli, Zaida Inês; Kuramae, Eiko Eurya; de Oliveira Camargo, Flávio Anastácio; Roesch, Luiz Fernando Würdig

    2013-01-01

    The Pampa biome is considered one of the main hotspots of the world’s biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated. PMID:24146873

  2. Soil-borne bacterial structure and diversity does not reflect community activity in Pampa biome.

    PubMed

    Lupatini, Manoeli; Suleiman, Afnan Khalil Ahmad; Jacques, Rodrigo Josemar Seminoti; Antoniolli, Zaida Inês; Kuramae, Eiko Eurya; de Oliveira Camargo, Flávio Anastácio; Roesch, Luiz Fernando Würdig

    2013-01-01

    The Pampa biome is considered one of the main hotspots of the world's biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated.

  3. Highly heterogeneous bacterial communities associated with the South China Sea reef corals Porites lutea, Galaxea fascicularis and Acropora millepora.

    PubMed

    Li, Jie; Chen, Qi; Zhang, Si; Huang, Hui; Yang, Jian; Tian, Xin-Peng; Long, Li-Juan

    2013-01-01

    Coral harbor diverse and specific bacteria play significant roles in coral holobiont function. Bacteria associated with three of the common and phylogenetically divergent reef-building corals in the South China Sea, Porites lutea, Galaxea fascicularis and Acropora millepora, were investigated using 454 barcoded-pyrosequencing. Three colonies of each species were sampled, and 16S rRNA gene libraries were constructed individually. Analysis of pyrosequencing libraries showed that bacterial communities associated with the three coral species were more diverse than previous estimates based on corals from the Caribbean Sea, Indo-Pacific reefs and the Red Sea. Three candidate phyla, including BRC1, OD1 and SR1, were found for the first time in corals. Bacterial communities were separated into three groups: P. lutea and G. fascicular, A. millepora and seawater. P. lutea and G. fascicular displayed more similar bacterial communities, and bacterial communities associated with A. millepora differed from the other two coral species. The three coral species shared only 22 OTUs, which were distributed in Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Chloroflexi, Actinobacteria, Acidobacteria and an unclassified bacterial group. The composition of bacterial communities within each colony of each coral species also showed variation. The relatively small common and large specific bacterial communities in these corals implies that bacterial associations may be structured by multiple factors at different scales and that corals may associate with microbes in terms of similar function, rather than identical species.

  4. Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

    PubMed Central

    Dembowski, Jill A.; Kuo, Benjamin; Woolford, John L.

    2013-01-01

    Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps. PMID:23788678

  5. Dietary and developmental shifts in butterfly-associated bacterial communities

    PubMed Central

    2018-01-01

    Bacterial communities associated with insects can substantially influence host ecology, evolution and behaviour. Host diet is a key factor that shapes bacterial communities, but the impact of dietary transitions across insect development is poorly understood. We analysed bacterial communities of 12 butterfly species across different developmental stages, using amplicon sequencing of the 16S rRNA gene. Butterfly larvae typically consume leaves of a single host plant, whereas adults are more generalist nectar feeders. Thus, we expected bacterial communities to vary substantially across butterfly development. Surprisingly, only few species showed significant dietary and developmental transitions in bacterial communities, suggesting weak impacts of dietary transitions across butterfly development. On the other hand, bacterial communities were strongly influenced by butterfly species and family identity, potentially due to dietary and physiological variation across the host phylogeny. Larvae of most butterfly species largely mirrored bacterial community composition of their diets, suggesting passive acquisition rather than active selection. Overall, our results suggest that although butterflies harbour distinct microbiomes across taxonomic groups and dietary guilds, the dramatic dietary shifts that occur during development do not impose strong selection to maintain distinct bacterial communities across all butterfly hosts. PMID:29892359

  6. Insight into the effects of different cropping systems on soil bacterial community and tobacco bacterial wilt rate.

    PubMed

    Niu, Jiaojiao; Chao, Jin; Xiao, Yunhua; Chen, Wu; Zhang, Chao; Liu, Xueduan; Rang, Zhongwen; Yin, Huaqun; Dai, Linjian

    2017-01-01

    Rotation is an effective strategy to control crop disease and improve plant health. However, the effects of crop rotation on soil bacterial community composition and structure, and crop health remain unclear. In this study, using 16S rRNA gene sequencing, we explored the soil bacterial communities under four different cropping systems, continuous tobacco cropping (control group), tobacco-maize rotation, tobacco-lily rotation, and tobacco-turnip rotation. Results of detrended correspondence analysis and dissimilarity tests showed that soil bacterial community composition and structure changed significantly among the four groups, such that Acidobacteria and Actinobacteria were more abundant in the maize rotation group (16.6 and 11.5%, respectively) than in the control (8.5 and 7.1%, respectively). Compared with the control group (57.78%), maize and lily were effective rotation crops in controlling tobacco bacterial wilt (about 23.54 and 48.67%). On the other hand, tobacco bacterial wilt rate was increased in the turnip rotation (59.62%) relative to the control. Further study revealed that the abundances of several bacterial populations were directly correlated with tobacco bacterial wilt. For example, Acidobacteria and Actinobacteria were significantly negatively correlated to the tobacco bacterial wilt rate, so they may be probiotic bacteria. Canonical correspondence analysis showed that soil pH and calcium content were key factors in determining soil bacterial communities. In conclusion, our study revealed the composition and structure of bacterial communities under four different cropping systems and may unveil molecular mechanisms for the interactions between soil microorganisms and crop health. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis.

    PubMed

    Zakaria, M N; Takeshita, T; Shibata, Y; Maeda, H; Wada, N; Akamine, A; Yamashita, Y

    2015-08-01

    To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  8. Microbial Diversity in Commercial Bee Pollen from Europe, Chile, and Mexico, Based on 16S rRNA Gene Amplicon Metagenome Sequencing.

    PubMed

    Moreno Andrade, Vicente D; Saldaña Gutiérrez, Carlos; Calvillo Medina, Rosa P; Cruz Hérnandez, Andrés; Vázquez Cruz, Moisés A; Torres Ruíz, Alfonso; Romero Gómez, Sergio; Ramos López, Miguel A; Álvarez-Hidalgo, Erika; López-Gaytan, Silvia B; Ramírez, Natanahel Salvador; Jones, George H; Hernandez-Flores, Jose Luis; Campos-Guillén, Juan

    2018-05-17

    Bee pollen is a highly nutritive natural foodstuff. Because of its use as a comestible, the association of bacteria with bee pollen is commercially and biologically important. We report here the bacterial diversity of seven bee pollen samples (five from Europe, one from Chile, and one from Mexico) based on 16S rRNA gene amplicon metagenome sequencing. Copyright © 2018 Moreno Andrade et al.

  9. Temperature effects on net greenhouse gas production and bacterial communities in arctic thaw ponds.

    PubMed

    Negandhi, Karita; Laurion, Isabelle; Lovejoy, Connie

    2016-08-01

    One consequence of High Arctic permafrost thawing is the formation of small ponds, which release greenhouse gases (GHG) from stored carbon through microbial activity. Under a climate with higher summer air temperatures and longer ice-free seasons, sediments of shallow ponds are likely to become warmer, which could influence enzyme kinetics or select for less cryophilic microbes. There is little data on the direct temperature effects on GHG production and consumption or on microbial communities' composition in Arctic ponds. We investigated GHG production over 16 days at 4°C and 9°C in sediments collected from four thaw ponds. Consistent with an enzymatic response, production rates of CO2 and CH4 were significantly greater at higher temperatures, with Q10 varying from 1.2 to 2.5. The bacterial community composition from one pond was followed through the incubation by targeting the V6-V8 variable regions of the 16S rRNA gene and 16S rRNA. Several rare taxa detected from rRNA accounted for significant community compositional changes. At the higher temperature, the relative community contribution from Bacteroidetes decreased by 15% with compensating increases in Betaproteobacteria, Alphaproteobacteria, Firmicutes, Acidobacteria, Verrucomicrobia and Actinobacteria. The increase in experimental GHG production accompanied by changes in community indicates an additional factor to consider in sediment environments when evaluating future climate scenarios. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goldfarb, K.C.; Karaoz, U.; Hanson, C.A.

    2011-04-18

    Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeledmore » DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin-protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial

  11. Comparison of Voltage Gated K+ Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

    PubMed

    Cox, Robert H; Fromme, Samantha

    2016-12-01

    We have shown that three components contribute to functional voltage gated K + (K v ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of K v subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K v subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

  12. Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene.

    PubMed

    da Mota, F F; Gomes, E A; Paiva, E; Rosado, A S; Seldin, L

    2004-01-01

    To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.

  13. Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

    PubMed Central

    Nishio, Kazuaki; Iwamoto-Kihara, Atsuko; Yamamoto, Akitsugu; Wada, Yoh; Futai, Masamitsu

    2002-01-01

    ATP synthase FoF1 (α3β3γδɛab2c10–14) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F1 β or membrane sector Fo a subunit when the Fo c subunit ring was immobilized; a biotin-tag was introduced onto the β or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the β or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the α or β subunit. These results established that the γɛc10–14 and α3β3δab2 complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis. PMID:12357031

  14. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring.

    PubMed

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

    2015-09-01

    The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche.

  15. Structure and dynamics of the bacterial communities in fermentation of the traditional Chinese post-fermented pu-erh tea revealed by 16S rRNA gene clone library.

    PubMed

    Zhao, Ming; Xiao, Wei; Ma, Yan; Sun, Tingting; Yuan, Wenxia; Tang, Na; Zhang, Donglian; Wang, Yongxia; Li, Yali; Zhou, Hongjie; Cui, Xiaolong

    2013-10-01

    Microbes are thought to have key roles in the development of the special properties of post-fermented pu-erh tea (pu-erh shucha), a well-known traditional Chinese tea; however, little is known about the bacteria during the fermentation. In this work, the structure and dynamics of the bacterial community involved in the production of pu-erh shucha were investigated using 16S rRNA gene clone libraries constructed from samples collected on days zero (LD-0), 5 (LD-5), 10 (LD-10), 15 (LD-15) and 20 (LD-20) of the fermentation. A total of 747 sequences with individual clone library containing 115-174 sequences and 4-20 unique operational taxonomic units (OTUs) were obtained. These OTUs were grouped into four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and further identified as members of 10 families, such as Alcaligenaceae, Bacillaceae, Enterobacteriaceae, etc. The dominant bacteria were Enterobacteriaceae in the raw material (LD-0) and in the initial stages of fermentation (LD-5 and LD-10), which changed to Bacillaceae at the last stages of fermentation (LD-15 and LD-20) at a temperature of 40-60 °C. It is interesting that the dominant OTUs in libraries LD-15 and LD-20 were very closely related to Bacillus coagulans, which is a safe thermoduric probiotic. Together the bacterial diversity and dynamics during a fermentation of pu-erh shucha were demonstrated, and a worthy clue for artificial inoculation of B. coagulans to improve the health benefits of pu-erh shucha or produce probiotic pu-erh tea were provided.

  16. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

    PubMed

    Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

    2015-11-01

    The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

    PubMed Central

    Vierheilig, J.; Savio, D.; Ley, R. E.; Mach, R. L.; Farnleitner, A. H.

    2016-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multicompartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  18. Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes.

    PubMed

    Segerstolpe, Asa; Lundkvist, Pär; Osheim, Yvonne N; Beyer, Ann L; Wieslander, Lars

    2008-08-01

    In Saccharomyces cerevisiae, synthesis of the small ribosomal subunit requires assembly of the 35S pre-rRNA into a 90S preribosomal complex. SnoRNAs, including U3 snoRNA, and many trans-acting proteins are required for the ordered assembly and function of the 90S preribosomal complex. Here, we show that the conserved protein Mrd1p binds to the pre-rRNA early during transcription and is required for compaction of the pre-18S rRNA into SSU processome particles. We have exploited the fact that an Mrd1p-GFP fusion protein is incorporated into the 90S preribosomal complex, where it acts as a partial loss-of-function mutation. When associated with the pre-rRNA, Mrd1p-GFP functionally interacts with the essential Pwp2, Mpp10 and U3 snoRNP subcomplexes that are functionally interconnected in the 90S preribosomal complex. The fusion protein can partially support 90S preribosome-mediated cleavages at the A(0)-A(2) sites. At the same time, on a substantial fraction of transcripts, the composition and/or structure of the 90S preribosomal complex is perturbed by the fusion protein in such a way that cleavage of the 35S pre-rRNA is either blocked or shifted to aberrant sites. These results show that Mrd1p is required for establishing productive structures within the 90S preribosomal complex.

  19. Culture-Independent Analysis of Indomethacin-Induced Alterations in the Rat Gastrointestinal Microbiota

    PubMed Central

    Dalby, Andrew B.; Frank, Daniel N.; St. Amand, Allison L.; Bendele, Alison M.; Pace, Norman R.

    2006-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for a variety of inflammatory conditions; however, the benefits of this class of drugs are accompanied by deleterious side effects, most commonly gastric irritation and ulceration. NSAID-induced ulceration is thought to be exacerbated by intestinal microbiota, but previous studies have not identified specific microbes that contribute to these adverse effects. In this study, we conducted a culture-independent analysis of ∼1,400 bacterial small-subunit rRNA genes associated with the small intestines and mesenteric lymph nodes of rats treated with the NSAID indomethacin. This is the first molecular analysis of the microbiota of the rat small intestine. A comparison of clone libraries and species-specific quantitative PCR results from rats treated with indomethacin and untreated rats revealed that organisms closely related to Enterococcus faecalis were heavily enriched in the small intestine and mesenteric lymph nodes of the treated rats. These data suggest that treatment of NSAID-induced ulceration may be facilitated by addressing the microbiological imbalances. PMID:17021222

  20. Onset and establishment of diazotrophs and other bacterial associates in the early life history stages of the coral Acropora millepora.

    PubMed

    Lema, Kimberley A; Bourne, David G; Willis, Bette L

    2014-10-01

    Early establishment of coral-microbial symbioses is fundamental to the fitness of corals, but comparatively little is known about the onset and succession of bacterial communities in their early life history stages. In this study, bacterial associates of the coral Acropora millepora were characterized throughout the first year of life, from larvae and 1-week-old juveniles reared in laboratory conditions in the absence of the dinoflagellate endosymbiont Symbiodinium to field-outplanted juveniles with established Symbiodinium symbioses, and sampled at 2 weeks and at 3, 6 and 12 months. Using an amplicon pyrosequencing approach, the diversity of both nitrogen-fixing bacteria and of bacterial communities overall was assessed through analysis of nifH and 16S rRNA genes, respectively. The consistent presence of sequences affiliated with diazotrophs of the order Rhizobiales (23-58% of retrieved nifH sequences; 2-12% of 16S rRNA sequences), across all samples from larvae to 12-month-old coral juveniles, highlights the likely functional importance of this nitrogen-fixing order to the coral holobiont. Dominance of Roseobacter-affiliated sequences (>55% of retrieved 16S rRNA sequences) in larvae and 1-week-old juveniles, and the consistent presence of sequences related to Oceanospirillales and Altermonadales throughout all early life history stages, signifies their potential importance as coral associates. Increased diversity of bacterial communities once juveniles were transferred to the field, particularly of Cyanobacteria and Deltaproteobacteria, demonstrates horizontal (environmental) uptake of coral-associated bacterial communities. Although overall bacterial communities were dynamic, bacteria with likely important functional roles remain stable throughout early life stages of Acropora millepora. © 2014 John Wiley & Sons Ltd.

  1. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DOE PAGES

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; ...

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy numbermore » of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.« less

  2. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    PubMed Central

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  3. A meta-barcoding approach to assess and compare the storage temperature-dependent bacterial diversity of gilt-head sea bream (Sparus aurata) originating from fish farms from two geographically distinct areas of Greece.

    PubMed

    Parlapani, Foteini F; Michailidou, Sofia; Pasentsis, Konstantinos; Argiriou, Anagnostis; Krey, Grigorios; Boziaris, Ioannis S

    2018-08-02

    Bacterial diversity of whole gilt-head sea bream (Sparus aurata L. 1758) originating from Ionian and Aegean Sea aquaculture farms and stored at 0 (ice), 4 and 8 °C was determined by 16S rRNA gene amplicon sequencing method using the Illumina's MiSeq platform. The composition of Aerobic Plate Counts (APC) was also monitored by 16S rRNA gene sequencing. The rejection time point of sea bream from either area, as determined by sensory evaluation, was about 14, 6 and 3 days at 0, 4 and 8 °C, respectively. APC was approximately 4.5 log cfu/g at day 0 and ranged from 7.5 to 8.5 log cfu/g at sensory rejection. Culture-depended analysis showed that Pseudomonas and Shewanella were the most abundant microorganisms grown on plates for both seas. Moreover, culture-independent analysis of DNA extracted directly from fish flesh showed that sea bream originating from different geographical areas exhibited different bacterial diversity. Pseudomonas and Psychrobacter were the dominant microorganisms of chill-stored fish from Ionian (apart from 8 °C, where Carnobacterium dominated) and Aegean Sea, respectively. In addition, small changes of storage temperature greatly affected bacterial microbiota of stored fish. Various bacterial species, not detected by conventional microbiological methods, were also revealed through 16S amplicon sequencing. In conclusion, the use of NGS approach is a promising methodology for assessing bacterial diversity of sea bream originating from different geographical areas and stored at various temperatures. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.

    No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newlymore » modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.« less

  5. Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

    PubMed

    Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

    2016-09-01

    Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

  6. The impact of persistent bacterial bronchitis on the pulmonary microbiome of children

    PubMed Central

    Bingle, Lynne; Cookson, William O. C. M.; Everard, Mark L.; Moffatt, Miriam F.

    2017-01-01

    Introduction Persistent bacterial bronchitis (PBB) is a leading cause of chronic wet cough in young children. This study aimed to characterise the respiratory bacterial microbiota of healthy children and to assess the impact of the changes associated with the development of PBB. Blind, protected brushings were obtained from 20 healthy controls and 24 children with PBB, with an additional directed sample obtained from PBB patients. DNA was extracted, quantified using a 16S rRNA gene quantitative PCR assay prior to microbial community analysis by 16S rRNA gene sequencing. Results No significant difference in bacterial diversity or community composition (R2 = 0.01, P = 0.36) was observed between paired blind and non-blind brushes, showing that blind brushings are a valid means of accessing the airway microbiota. This has important implications for collecting lower respiratory samples from healthy children. A significant decrease in bacterial diversity (P < 0.001) and change in community composition (R2 = 0.08, P = 0.004) was observed among controls, in comparison with patients. Bacterial communities within patients with PBB were dominated by Proteobacteria, and indicator species analysis showed that Haemophilus and Neisseria were significantly associated with the patient group. In 15 (52.9%) cases the dominant organism by sequencing was not identified by standard routine clinical culture. Conclusion The bacteria present in the lungs of patients with PBB were less diverse in terms of richness and evenness. The results validate the clinical diagnosis, and suggest that more attention to bacterial communities in children with chronic cough may lead to more rapid recognition of this condition with earlier treatment and reduction in disease burden. PMID:29281698

  7. Small Subunits of Serine Palmitoyltransferase (ssSPTs) and Their Physiological Roles

    DTIC Science & Technology

    2014-02-12

    showing that organisms also have unique sphingoid base chain lengths. In insects, such as Drosophila melanogaster , the predominant chain lengths of the ... Drosophila melanogaster mutant defective in male meiotic cytokinesis (‘Ghiberti’) has a mutation in a gene with low homology to the ssSPT subunits of...INTRODUCTION: Sphingolipid metabolism in Drosophila melanogaster (fly) is an active area of research. It is a good model system to study the roles of

  8. Description of Drinking Water Bacterial Communities Using 16S rRNA Gene Sequence Analyses

    EPA Science Inventory

    Descriptions of bacterial communities inhabiting water distribution systems (WDS) have mainly been accomplished using culture-based approaches. Due to the inherent selective nature of culture-based approaches, the majority of bacteria inhabiting WDS remain uncharacterized. The go...

  9. Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

    PubMed

    Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2013-06-14

    Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

  10. What tests should you use to assess small intestinal bacterial overgrowth in systemic sclerosis?

    PubMed

    Braun-Moscovici, Yolanda; Braun, Marius; Khanna, Dinesh; Balbir-Gurman, Alexandra; Furst, Daniel E

    2015-01-01

    Small intestinal bacterial overgrowth (SIBO) plays a major role in the pathogenesis of malabsorption in SSc patients and is a source of great morbidity and even mortality, in those patients. This manuscript reviews which tests are valid and should be used in SSc when evaluating SIBO. We performed systematic literature searches in PubMed, Embase and the Cochrane library from 1966 up to November 2014 for English language, published articles examining bacterial overgrowth in SSc (e.g. malabsorption tests, breath tests, xylose test, etc). Articles obtained from these searches were reviewed for additional references. The validity of the tests was evaluated according to the OMERACT principles of truth, discrimination and feasibility. From a total of 65 titles, 22 articles were reviewed and 20 were ultimately extracted to examine the validity of tests for GI morphology, bacterial overgrowth and malabsorption in SSc. Only 1 test (hydrogen and methane breath tests) is fully validated. Four tests are partially validated, including jejunal cultures, xylose, lactulose tests, and 72 hours fecal fat test. Only 1 of a total of 5 GI tests of bacterial overgrowth (see above) is fully validated in SSc. For clinical trials, fully validated tests are preferred, although some investigators use partially validated tests (4 tests). Further validation of GI tests in SSc is needed.

  11. Bacterial communities in floral nectar.

    PubMed

    Fridman, Svetlana; Izhaki, Ido; Gerchman, Yoram; Halpern, Malka

    2012-02-01

    Floral nectar is regarded as the most important reward available to animal-pollinated plants to attract pollinators. Despite the vast amount of publications on nectar properties, the role of nectar as a natural bacterial habitat is yet unexplored. To gain a better understanding of bacterial communities inhabiting floral nectar, culture-dependent and -independent (454-pyrosequencing) methods were used. Our findings demonstrate that bacterial communities in nectar are abundant and diverse. Using culture-dependent method we showed that bacterial communities of nectar displayed significant variation among three plant species: Amygdalus communis, Citrus paradisi and Nicotiana glauca. The dominant class in the nectar bacterial communities was Gammaproteobacteria. About half of the isolates were novel species (< 97% similarities of the 16S rRNA gene with known species). Using 454-pyrosequencing we demonstrated that nectar microbial community are distinct for each of the plant species while there are no significant differences between nectar microbial communities within nectars taken from different plants of the same species. Primary selection of the nectar bacteria is unclear; it may be affected by variations in the chemical composition of the nectar in each plant. The role of the rich and diverse nectar microflora in the attraction-repulsion relationships between the plant and its nectar consumers has yet to be explored. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  12. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    PubMed

    McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  13. Peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit

    PubMed Central

    Bian, Xiaochun; Ren, Jianhua; De Vries, Matthew; Schnegelsberg, Birthe; Cockayne, Debra A; Ford, Anthony P D W; Galligan, James J

    2003-01-01

    P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. P2X receptors participate in intestinal neurotransmission but the subunit composition of enteric P2X receptors is unknown. In this study, we used tissues from P2X3 wild-type (P2X3+/+) mice and mice in which the P2X3 subunit gene had been deleted (P2X3−/−) to investigate the role of this subunit in neurotransmission in the intestine. RT-PCR analysis of mRNA from intestinal tissues verified P2X3 gene deletion. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from myenteric neurons in vitro. Drug-induced longitudinal muscle contractions were studied in vitro. Intraluminal pressure-induced reflex contractions (peristalsis) of ileal segments were studied in vitro using a modified Trendelenburg preparation. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. Fast excitatory postsynaptic potentials recorded from S neurons (motoneurons and interneurons) were similar in tissues from P2X3+/+ and P2X3−/− mice. S neurons from P2X3+/+ and P2X3−/− mice were depolarized by application of ATP but not α,β-methylene ATP, an agonist of P2X3 subunit-containing receptors. ATP and α,β-methylene ATP induced depolarization of AH (sensory) neurons from P2X3+/+ mice. ATP, but not α,β-methylene ATP, caused depolarization of AH neurons from P2X3−/− mice. Peristalsis was inhibited in ileal segments from P2X3−/− mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X3+/+ and P2X3−/− mice. Gastrointestinal transit was similar in P2X3+/+ and P2X3−/− mice. It is concluded that P2X3 subunit-containing receptors participate in neural pathways underlying peristalsis in the mouse intestine in vitro. P2X3 subunits are localized to AH (sensory) but not S neurons. P2X3 receptors may

  14. Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

    PubMed Central

    Umaer, Khan; Ciganda, Martin

    2014-01-01

    Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention. PMID:24706018

  15. Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. I. Transmission process.

    PubMed

    Gómez-Gutiérrez, Jaime; López-Cortés, Alejandro; Aguilar-Méndez, Mario J; Del Angel-Rodríguez, Jorge A; Tremblay, Nelly; Zenteno-Savín, Tania; Robinson, Carlos J

    2015-10-27

    Histophagous ciliates of the genus Pseudocollinia cause epizootic events that kill adult female krill (Euphausiacea), but their mode of transmission is unknown. We compared 16S rRNA sequences of bacterial strains isolated from stomachs of healthy krill Nyctiphanes simplex specimens with sequences of bacterial isolates and sequences of natural bacterial communities from the hemocoel of N. simplex specimens infected with P. brintoni to determine possible transmission pathways. All P. brintoni endoparasitic life stages and the transmission tomite stage (outside the host) were associated with bacterial assemblages. 16S rRNA sequences from isolated bacterial strains showed that Photobacterium spp. and Pseudoalteromonas spp. were dominant members of the bacterial assemblages during all life phases of P. brintoni and potential pathobionts. They were apparently unaffected by the krill's immune system or the histophagous activity of P. brintoni. However, other bacterial strains were found only in certain P. brintoni life phases, indicating that as the infection progressed, microhabitat conditions and microbial interactions may have become unfavorable for some strains of bacteria. Trophic infection is the most parsimonious explanation for how P. brintoni infects krill. We estimated N. simplex vulnerability to P. brintoni infection during more than three-fourths of their life span, infecting mostly adult females. The ciliates have relatively high prevalence levels (albeit at <10% of sampled stations) and a short life cycle (estimated <7 d). Histophagous ciliate-krill interactions may occur in other krill species, particularly those that form dense swarms and attain high population densities that potentially enhance trophic transmission and allow completion of the Pseudocollinia spp. life cycle.

  16. Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning.

    PubMed

    Zhao, Zhongliang; Dammert, Marcel A; Hoppe, Sven; Bierhoff, Holger; Grummt, Ingrid

    2016-09-30

    Attenuation of ribosome biogenesis in suboptimal growth environments is crucial for cellular homeostasis and genetic integrity. Here, we show that shutdown of rRNA synthesis in response to elevated temperature is brought about by mechanisms that target both the RNA polymerase I (Pol I) transcription machinery and the epigenetic signature of the rDNA promoter. Upon heat shock, the basal transcription factor TIF-IA is inactivated by inhibition of CK2-dependent phosphorylations at Ser170/172. Attenuation of pre-rRNA synthesis in response to heat stress is accompanied by upregulation of PAPAS, a long non-coding RNA (lncRNA) that is transcribed in antisense orientation to pre-rRNA. PAPAS interacts with CHD4, the adenosine triphosphatase subunit of NuRD, leading to deacetylation of histones and movement of the promoter-bound nucleosome into a position that is refractory to transcription initiation. The results exemplify how stress-induced inactivation of TIF-IA and lncRNA-dependent changes of chromatin structure ensure repression of rRNA synthesis in response to thermo-stress. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Characterizing bacterial communities in tilapia pond surface sediment and their responses to pond differences and temporal variations.

    PubMed

    Fan, Limin; Barry, Kamira; Hu, Gengdong; Meng, Shunlong; Song, Chao; Qiu, Liping; Zheng, Yao; Wu, Wei; Qu, Jianhong; Chen, Jiazhang; Xu, Pao

    2017-01-01

    Bacterial community compositions in the surface sediment of tilapia ponds and their responses to pond characteristics or seasonal variations were investigated. For that, three ponds with different stocking densities were selected to collect the samples. And the method of Illumina high-throughput sequencing was used to amplify the bacterial 16S rRNA genes. A total of 662, 876 valid reads and 5649 operational taxonomic units were obtained. Further analysis showed that the dominant phyla in all three ponds were Proteobacteria, Bacteroidetes, Chloroflexi, and Acidobacteria. The phyla Planctomycetes, Firmicutes, Chlorobi, and Spirochaetae were also relatively abundant. Among the eight phyla, the abundances of only Proteobacteria, Bacteroidetes, Firmicutes, and Spirochaetae were affected by seasonal variations, while seven of these (with the exception of Acidobacteria) were affected by pond differences. A comprehensive analysis of the richness and diversity of the bacterial 16S rRNA gene, and of the similarity in bacterial community composition in sediment also showed that the communities in tilapia pond sediment were shaped more by pond differences than by seasonal variations. Linear discriminant analysis further indicated that the influences of pond characteristics on sediment bacterial communities might be related to feed coefficients and stocking densities of genetically improved farmed tilapia (GIFT).

  18. Microbial profiles of a drinking water resource based on different 16S rRNA V regions during a heavy cyanobacterial bloom in Lake Taihu, China.

    PubMed

    Zhang, Junyi; Zhu, Congming; Guan, Rui; Xiong, Zhipeng; Zhang, Wen; Shi, Junzhe; Sheng, Yi; Zhu, Bingchuan; Tu, Jing; Ge, Qinyu; Chen, Ting; Lu, Zuhong

    2017-05-01

    Understanding of the bacterial community structure in drinking water resources helps to enhance the security of municipal water supplies. In this study, bacterial communities were surveyed in water and sediment during a heavy cyanobacterial bloom in a drinking water resource of Lake Taihu, China. A total of 325,317 high-quality sequences were obtained from different 16S ribosomal RNA (rRNA) regions (V3, V4, and V6) using the Miseq sequencing platform. A notable difference was shown between the water and sediment samples, as predominated by Cyanobacteria, Proteobacteria, and Actinobacteria in the water and Proteobacteria, Chloroflexi, and Verrucomicrobia in the sediment, respectively. The LD12 family dominated the water surface and was tightly associated with related indicators of cyanobacterial propagation, indicating involvement in the massive proliferation of cyanobacterial blooms. Alternatively, the genus Nitrospira dominated the sediment samples, which indicates that nitrite oxidation was very active in the sediment. Although pathogenic bacteria were not detected in a large amount, some genera such as Mycobacterium, Acinetobacter, and Legionella were still identified but in very low abundance. In addition, the effects of different V regions on bacterial diversity survey were evaluated. Overall, V4 and V3 were proven to be more promising V regions for bacterial diversity survey in water and sediment samples during heavy water blooms in Lake Taihu, respectively. As longer, cheaper, and faster DNA sequencing technologies become more accessible, we expect that bacterial community structures based on 16S rRNA amplicons as an indicator could be used alongside with physical and chemical indicators, to conduct comprehensive assessments for drinking water resource management.

  19. Exploring the bacterial gut microbiota of supralittoral talitrid amphipods.

    PubMed

    Abdelrhman, Khaled F A; Bacci, Giovanni; Marras, Barbara; Nistri, Annamaria; Schintu, Marco; Ugolini, Alberto; Mengoni, Alessio

    2017-01-01

    Talitrid amphipods (sandhoppers and beach fleas) are typical of the supralittoral zone. They are known to thrive on stranded materials, including detrital marine angiosperms and macroalgae, as well as occasional dead animals. In this work, the gut microbiota of five species of talitrid amphipods (Talitrus saltator, Talorchestia ugolinii, Sardorchestia pelecaniformis, Orchestia montagui and Orchestia stephenseni) collected in Sardinia (Italy) has been investigated through: i) metabarcoding analysis of the amplified 16S rRNA V4 region; and ii) quantification of family 48 glycosyl hydrolase genes (GHF48), involved in cellulose degradation. Results indicate that, though talitrid gut biodiversity is not directly related to taxon or sampling locality, the animals' digestive tracts may host species-specific bacterial communities. In particular, gut microbiota of O. montagui, an inhabitant of Posidonia banquettes and macro-algae mat, showed the greatest differences in taxonomic composition and the highest proportion of GHF48 genes with respect to 16S rRNA genes. These results suggest that the different talitrid species may host species-specific bacterial communities whose function could partially reflect the different microhabitats and food preferences of their host. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring

    PubMed Central

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

    2015-01-01

    The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche. PMID:26484255

  1. Cloning and characterization of two novel zebrafish P2X receptor subunits.

    PubMed

    Diaz-Hernandez, Miguel; Cox, Jane A; Migita, Keisuke; Haines, William; Egan, Terrance M; Voigt, Mark M

    2002-07-26

    In this report we describe the cloning and characterization of two P2X receptor subunits cloned from the zebrafish (Danio rerio). Primary sequence analysis suggests that one cDNA encodes an ortholog of the mammalian P2X(4) subunit and the second cDNA encodes the ortholog of the mammalian P2X(5) subunit. The zP2X(4) subunit forms a homo-oligomeric receptor that displays a low affinity for ATP (EC(50)=274+/-48 microM) and very low affinity (EC(50)>500 microM) for other purinergic ligands such as alphabetameATP, suramin, and PPADS. As seen with the mammalian orthologs, the zP2X(5) subunit forms a homo-oligomeric receptor that yields very small whole-cell currents (<20pA), making determination of an EC(50) problematic. Both subunit genes were physically mapped onto the zebrafish genome using radiation hybrid analysis of the T51 panel, with the zp2x4 localized to LG21 and zp2x5 to LG5.

  2. Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides.

    PubMed

    Chopyk, Jessica; Moore, Ryan M; DiSpirito, Zachary; Stromberg, Zachary R; Lewis, Gentry L; Renter, David G; Cernicchiaro, Natalia; Moxley, Rodney A; Wommack, K Eric

    2016-03-22

    Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide

  3. Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

    PubMed Central

    Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

    1988-01-01

    A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

  4. Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

    PubMed

    Nascimento, Diana S; do Vale, Ana; Tomás, Ana M; Zou, Jun; Secombes, Christopher J; dos Santos, Nuno M S

    2007-03-01

    Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

  5. Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

    PubMed Central

    Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro

    2014-01-01

    Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na+/H+ antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na+. PMID:24142251

  6. Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.

    PubMed

    Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro; Krulwich, Terry Ann

    2014-01-01

    Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

  7. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    PubMed

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  8. The gamma subunit of transducin is farnesylated.

    PubMed Central

    Lai, R K; Perez-Sala, D; Cañada, F J; Rando, R R

    1990-01-01

    Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification. Images PMID:2217200

  9. Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism.

    PubMed Central

    Fernandes, A P; Nelson, K; Beverley, S M

    1993-01-01

    Molecular evolutionary relationships within the protozoan order Kinetoplastida were deduced from comparisons of the nuclear small and large subunit ribosomal RNA (rRNA) gene sequences. These studies show that relationships among the trypanosomatid protozoans differ from those previously proposed from studies of organismal characteristics or mitochondrial rRNAs. The genera Leishmania, Endotrypanum, Leptomonas, and Crithidia form a closely related group, which shows progressively more distant relationships to Phytomonas and Blastocrithidia, Trypanosoma cruzi, and lastly Trypanosoma brucei. The rooting of the trypanosomatid tree was accomplished by using Bodo caudatus (family Bodonidae) as an outgroup, a status confirmed by molecular comparisons with other eukaryotes. The nuclear rRNA tree agrees well with data obtained from comparisons of other nuclear genes. Differences with the proposed mitochondrial rRNA tree probably reflect the lack of a suitable outgroup for this tree, as the topologies are otherwise similar. Small subunit rRNA divergences within the trypanosomatids are large, approaching those among plants and animals, which underscores the evolutionary antiquity of the group. Analysis of the distribution of different parasitic life-styles of these species in conjunction with a probable timing of evolutionary divergences suggests that vertebrate parasitism arose multiple times in the trypanosomatids. PMID:8265597

  10. Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

    PubMed

    Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

    2009-03-01

    Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

  11. The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

    PubMed Central

    Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

    2010-01-01

    Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

  12. Pyrosequencing of Bacterial Symbionts within Axinella corrugata Sponges: Diversity and Seasonal Variability

    PubMed Central

    White, James R.; Patel, Jignasa; Ottesen, Andrea; Arce, Gabriela; Blackwelder, Patricia; Lopez, Jose V.

    2012-01-01

    Background Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge “holobiont” system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. Methodology/Principal Findings We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs). Approximately 65,550 rRNA sequences (24%) could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa), and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising >34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. Conclusions/Significance Slight shifts in several bacterial taxa were observed

  13. Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

    PubMed Central

    McLachlan, Sandra M.; Aliesky, Holly A.; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  14. A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic

    PubMed Central

    2017-01-01

    The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB. PMID:28154188

  15. Bacterial and fungal core microbiomes associated with small grain silages during ensiling and aerobic spoilage.

    PubMed

    Duniere, Lysiane; Xu, Shanwei; Long, Jin; Elekwachi, Chijioke; Wang, Yuxi; Turkington, Kelly; Forster, Robert; McAllister, Tim A

    2017-03-03

    Describing the microbial populations present in small grain silage and understanding their changes during ensiling is of interest for improving the nutrient value of these important forage crops. Barley, oat and triticale forages as well as an intercropped mixture of the 3 crops were harvested and ensiled in mini silos for a period of 90 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics and nutritive value were assessed in terminal silages and bacterial and fungal communities during ensiling and aerobic exposure were described using 16S and 18S rDNA sequencing, respectively. All small grain silages exhibited chemical traits that were associated with well ensiled forages, such as low pH value (4.09 ± 0.28) and high levels of lactic acid (59.8 ± 14.59 mg/g DM). The number of microbial core genome operational taxonomic units (OTUs) decreased with time of ensiling. Taxonomic bacterial community profiles were dominated by the Lactobacillales after fermentation, with a notable increase in Bacillales as a result of aerobic exposure. Diversity of the fungal core microbiome was shown to also be reduced during ensiling. Operational taxonomic units assigned to filamentous fungi were found in the core microbiome at ensiling and after aerobic exposure, whereas the Saccharomycetales were the dominate yeast population after 90 days of ensiling and aerobic exposure. Bacterial and fungal orders typically associated with silage spoilage were identified in the core microbiome after aerobic exposure. Next Generation Sequencing was successfully used to describe bacterial communities and the first record of fungal communities throughout the process of ensiling and utilization. Adequately describing the microbial ecology of silages could lead to improved ensiling practices and the selection of silage inoculants that act synergistically with the natural forage microbiome.

  16. Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings.

    PubMed

    Schabereiter-Gurtner, C; Piñar, G; Vybiral, D; Lubitz, W; Rölleke, S

    2001-11-01

    A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.

  17. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

    NASA Technical Reports Server (NTRS)

    Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

  18. Systematic Review and Meta-Analysis: Prevalence of Small Intestinal Bacterial Overgrowth in Chronic Liver Disease.

    PubMed

    Shah, Ayesha; Shanahan, Erin; Macdonald, Graeme A; Fletcher, Linda; Ghasemi, Pegah; Morrison, Mark; Jones, Mike; Holtmann, Gerald

    2017-11-01

    The authors conducted a meta-analysis of the prevalence of small intestinal bacterial overgrowth (SIBO) in patients with chronic liver disease (CLD) and controls. Using the search terms "small intestinal bacterial overgrowth (SIBO)" and "chronic liver disease (CLD)" or "cirrhosis," 19 case-control studies were identified. Utilizing breath tests, the prevalence of SIBO in CLD was 35.80% (95% CI, 32.60-39.10) compared with 8.0% (95% CI, 5.70-11.00) in controls. Using culture techniques, the prevalence was 68.31% (95% CI, 59.62-76.00) in CLD patients as compared with 7.94% (95% CI, 3.44-12.73) in controls. No difference between cirrhotic and noncirrhotic patients was found. SIBO is significantly more frequent in CLD patients as compared with controls. The association of SIBO and CLD was not confined to patients with advanced CLD, suggesting that SIBO is not a consequence of advanced liver disease but may play a role in the progression of CLD. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  19. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence TimeS⃞

    PubMed Central

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J. G.

    2011-01-01

    No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo. PMID:21890748

  20. [Analysis of bacterial small-colony variants isolated from clinical specimens].

    PubMed

    Matsumoto, Takehisa

    2014-07-01

    There is a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic traits called bacterial small-colony variants (SCVs). Phenotypically, SCVs show a slow growth rate, atypical colony morphology, and unusual biochemical characteristics. SCV strains often grow on blood agar or Drigalski agar as non-pigmented or pinpoint pigmented colonies, and key biochemical tests for them are often non-reactive. This review describes analyses of hemin-dependent Escherichia coli SCV and Staphylococcus aureus thymidine-dependent SCVs based on our case reports. Because SCVs exhibit fastidious growth characteristics, clinical microbiologists may easily miss or misidentify them in the clinical laboratory. Therefore, we must elucidate the cause of SCVs, and improve laboratory methods for the identification and assessment of the susceptibility of SCVs in the clinical laboratory.