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Sample records for escherichia coli chromosome

  1. Profiling of Escherichia coli Chromosome database.

    PubMed

    Yamazaki, Yukiko; Niki, Hironori; Kato, Jun-ichi

    2008-01-01

    The Profiling of Escherichia coli Chromosome (PEC) database (http://www.shigen.nig.ac.jp/ecoli/pec/) is designed to allow E. coli researchers to efficiently access information from functional genomics studies. The database contains two principal types of data: gene essentiality and a large collection of E. coli genetic research resources. The essentiality data are based on data compilation from published single-gene essentiality studies and on cell growth studies of large-deletion mutants. Using the circular and linear viewers for both whole genomes and the minimal genome, users can not only gain an overview of the genome structure but also retrieve information on contigs, gene products, mutants, deletions, and so forth. In particular, genome-wide exhaustive mutants are an essential resource for studying E. coli gene functions. Although the genomic database was constructed independently from the genetic resources database, users may seamlessly access both types of data. In addition to these data, the PEC database also provides a summary of homologous genes of other bacterial genomes and of protein structure information, with a comprehensive interface. The PEC is thus a convenient and useful platform for contemporary E. coli researchers.

  2. Dynamic organization of chromosomal DNA in Escherichia coli.

    PubMed

    Niki, H; Yamaichi, Y; Hiraga, S

    2000-01-15

    We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.

  3. Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics.

    PubMed

    Cass, Julie A; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2016-06-21

    The structure of the Escherichia coli chromosome is inherently dynamic over the duration of the cell cycle. Genetic loci undergo both stochastic motion around their initial positions and directed motion to opposite poles of the rod-shaped cell during segregation. We developed a quantitative method to characterize cell-cycle dynamics of the E. coli chromosome to probe the chromosomal steady-state mobility and segregation process. By tracking fluorescently labeled chromosomal loci in thousands of cells throughout the entire cell cycle, our method allows for the statistical analysis of locus position and motion, the step-size distribution for movement during segregation, and the locus drift velocity. The robust statistics of our detailed analysis of the wild-type E. coli nucleoid allow us to observe loci moving toward midcell before segregation occurs, consistent with a replication factory model. Then, as segregation initiates, we perform a detailed characterization of the average segregation velocity of loci. Contrary to origin-centric models of segregation, which predict distinct dynamics for oriC-proximal versus oriC-distal loci, we find that the dynamics of loci were universal and independent of genetic position.

  4. Chromosome segregation by the Escherichia coli Min system

    PubMed Central

    Di Ventura, Barbara; Knecht, Benoît; Andreas, Helena; Godinez, William J; Fritsche, Miriam; Rohr, Karl; Nickel, Walter; Heermann, Dieter W; Sourjik, Victor

    2013-01-01

    The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane-associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP-dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology. PMID:24022004

  5. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

    PubMed

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili; Christensen, Jens P; Olsen, John E; Nolan, Lisa; Olsen, Rikke H

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli.

  6. Coordination between chromosome replication and cell division in Escherichia coli.

    PubMed Central

    Tang, M S; Helmstetter, C E

    1980-01-01

    Cell division properties of Escherichia coli B/r containing either a dnaC or a dnaI mutation were examined. Incubation at nonpermissive temperature resulted in the eventual production of cells of approximately normal size, or slightly smaller, which lacked chromosomal DNA. The cell division patterns in cultures which were grown at permissive temperature and then shifted to nonpermissive temperature were consistent with: first, division and equipartition of chromosomes by cells which were in the C and D periods at the time of the shift; second, an apparent delay in cell division; and third, commencement of the formation of chromosomeless cells. In glucose-grown cultures of the dnaI mutant, production of chromosomeless cells continued for at least 120 min, whereas in the dnaC mutant chromosomeless cells were formed during a single interval between 110 and 130 min after the temperature shift. The results are discussed in light of the hypothesis that replication of a specific chromosomal region is not an obligatory requirement for the initiation and completion of the processes leading to division in a cell which contains at least one functioning chromosome. PMID:6988405

  7. Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli

    PubMed Central

    Fingland, Nicholas; Flåtten, Ingvild; Downey, Christopher D; Fossum-Raunehaug, Solveig; Skarstad, Kirsten; Crooke, Elliott

    2012-01-01

    In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication. We observe here that when acidic phospholipids were depleted, replication was inhibited with a concomitant reduction of chromosomal content and cell mass prior to growth arrest. This global shutdown of biosynthetic activity was independent of the stringent response. Restoration of acidic phospholipid synthesis resulted in a resumption of DNA replication prior to restored growth, indicating a possible cell-cycle-specific growth arrest had occurred with the earlier loss of acidic phospholipids. Flow cytometry, thymidine uptake, and quantitative polymerase chain reaction data suggest that a deficiency in acidic phospholipids prolonged the time required to replicate the chromosome. We also observed that regardless of the cellular content of acidic phospholipids, expression of mutant DnaA(L366K) altered the DNA content-to-cell mass ratio. PMID:23233230

  8. Nucleotide sequence of an Escherichia coli chromosomal hemolysin.

    PubMed Central

    Felmlee, T; Pellett, S; Welch, R A

    1985-01-01

    We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented. Images PMID:3891743

  9. Molecular Evolution of the Escherichia Coli Chromosome. IV. Sequence Comparisons

    PubMed Central

    Milkman, R.; Bridges, M. M.

    1993-01-01

    DNA sequences have been compared in a 4,400-bp region for Escherichia coli K12 and 36 ECOR strains. Discontinuities in degree of similarity, previously inferred, are confirmed in detail. Three clonal frames are described on the basis of the present local high-resolution data, as well as previous analyses of restriction fragment length polymorphism (RFLP) and of multilocus enzyme electrophoresis (MLEE) covering small regions more widely dispersed on the chromosome. These three approaches show important consistency. The data illustrate the fact that, in the limited context of intraspecific genomic sequence variation, clonality and homology are synonymous. Two estimable quantitative properties are defined: recency of common ancestry (the reciprocal of the log(10) of the number of generations since the most recent common ancestor), and the number of nucleotide pairs over which a given recency of common ancestry applies. In principle, these parameters are measures of the degree and physical extent of homology. The small size of apparent recombinational replacements, together with the observation that they occasionally occur in discontinuous series, raises the question of whether they result from the superimposition of replacements of much larger size (as expected from an elementary interpretation of conjugation and transduction in experimental E. coli systems) or via an alternative mechanism. Length polymorphisms of several sorts are described. PMID:8095913

  10. Chromosome Replication in Escherichia coli: Life on the Scales

    PubMed Central

    Norris, Vic; Amar, Patrick

    2012-01-01

    At all levels of Life, systems evolve on the 'scales of equilibria'. At the level of bacteria, the individual cell must favor one of two opposing strategies and either take risks to grow or avoid risks to survive. It has been proposed in the Dualism hypothesis that the growth and survival strategies depend on non-equilibrium and equilibrium hyperstructures, respectively. It has been further proposed that the cell cycle itself is the way cells manage to balance the ratios of these types of hyperstructure so as to achieve the compromise solution of living on the two scales. Here, we attempt to re-interpret a major event, the initiation of chromosome replication in Escherichia coli, in the light of scales of equilibria. This entails thinking in terms of hyperstructures as responsible for intensity sensing and quantity sensing and how this sensing might help explain the role of the DnaA protein in initiation of replication. We outline experiments and an automaton approach to the cell cycle that should test and refine the scales concept. PMID:25371267

  11. Characterization of pyridoxine auxotrophs of Escherichia coli: chromosomal position of linkage group I.

    PubMed

    Dempsey, W B

    1969-10-01

    The chromosomal location of Group I pyridoxine mutations in Escherichia coli is shown to be adjacent to dsdA,aroC, and purF (old purC) in E. coli B x K-12 hybrids. All mutants previously classified into Group I by nutrition tests and transduction frequency tests are shown to be linked to dsdA.

  12. Characterization of Pyridoxine Auxotrophs of Escherichia coli: Chromosomal Position of Linkage Group I

    PubMed Central

    Dempsey, Walter B.

    1969-01-01

    The chromosomal location of Group I pyridoxine mutations in Escherichia coli is shown to be adjacent to dsdA,aroC, and purF (old purC) in E. coli B × K-12 hybrids. All mutants previously classified into Group I by nutrition tests and transduction frequency tests are shown to be linked to dsdA. PMID:4898994

  13. Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae.

    PubMed

    Messerschmidt, Sonja J; Kemter, Franziska S; Schindler, Daniel; Waldminghaus, Torsten

    2015-02-01

    Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli.

    PubMed

    Galli, Elisa; Midonet, Caroline; Paly, Evelyne; Barre, François-Xavier

    2017-03-01

    Homologous recombination between the circular chromosomes of bacteria can generate chromosome dimers. They are resolved by a recombination event at a specific site in the replication terminus of chromosomes, dif, by dedicated tyrosine recombinases. The reaction is under the control of a cell division protein, FtsK, which assembles into active DNA pumps at mid-cell during septum formation. Previous studies suggested that activation of Xer recombination at dif was restricted to chromosome dimers in Escherichia coli but not in Vibrio cholerae, suggesting that FtsK mainly acted on chromosome dimers in E. coli but frequently processed monomeric chromosomes in V. cholerae. However, recent microscopic studies suggested that E. coli FtsK served to release the MatP-mediated cohesion and/or cell division apparatus-interaction of sister copies of the dif region independently of chromosome dimer formation. Here, we show that these apparently paradoxical observations are not linked to any difference in the dimer resolution machineries of E. coli and V. cholerae but to differences in the timing of segregation of their chromosomes. V. cholerae harbours two circular chromosomes, chr1 and chr2. We found that whatever the growth conditions, sister copies of the V. cholerae chr1 dif region remain together at mid-cell until the onset of constriction, which permits their processing by FtsK and the activation of dif-recombination. Likewise, sister copies of the dif region of the E. coli chromosome only separate after the onset of constriction in slow growth conditions. However, under fast growth conditions the dif sites separate before constriction, which restricts XerCD-dif activity to resolving chromosome dimers.

  15. Optimization and Characterization of the Synthetic Secondary Chromosome synVicII in Escherichia coli

    PubMed Central

    Messerschmidt, Sonja J.; Schindler, Daniel; Zumkeller, Celine M.; Kemter, Franziska S.; Schallopp, Nadine; Waldminghaus, Torsten

    2016-01-01

    Learning by building is one of the core ideas of synthetic biology research. Consequently, building synthetic chromosomes is the way to fully understand chromosome characteristics. The last years have seen exciting synthetic chromosome studies. We had previously introduced the synthetic secondary chromosome synVicII in Escherichia coli. It is based on the replication mechanism of the secondary chromosome in Vibrio cholerae. Here, we present a detailed analysis of its genetic characteristics and a selection approach to optimize replicon stability. We probe the origin diversity of secondary chromosomes from Vibrionaceae by construction of several new respective replicons. Finally, we present a synVicII version 2.0 with several innovations including its full compatibility with the popular modular cloning (MoClo) assembly system. PMID:28066763

  16. The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome.

    PubMed

    Gumbiner-Russo, L M; Lombardo, M J; Ponder, R G; Rosenberg, S M

    2001-07-25

    Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.

  17. Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

    PubMed Central

    Juhas, Mario; Ajioka, James W.

    2015-01-01

    Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci. PMID:25816013

  18. Persistent super-diffusive motion of Escherichia coli chromosomal loci.

    PubMed

    Javer, Avelino; Kuwada, Nathan J; Long, Zhicheng; Benza, Vincenzo G; Dorfman, Kevin D; Wiggins, Paul A; Cicuta, Pietro; Lagomarsino, Marco Cosentino

    2014-05-30

    The physical nature of the bacterial chromosome has important implications for its function. Using high-resolution dynamic tracking, we observe the existence of rare but ubiquitous 'rapid movements' of chromosomal loci exhibiting near-ballistic dynamics. This suggests that these movements are either driven by an active machinery or part of stress-relaxation mechanisms. Comparison with a null physical model for subdiffusive chromosomal dynamics shows that rapid movements are excursions from a basal subdiffusive dynamics, likely due to driven and/or stress-relaxation motion. Additionally, rapid movements are in some cases coupled with known transitions of chromosomal segregation. They do not co-occur strictly with replication, their frequency varies with growth condition and chromosomal coordinate, and they show a preference for longitudinal motion. These findings support an emerging picture of the bacterial chromosome as off-equilibrium active matter and help developing a correct physical model of its in vivo dynamic structure.

  19. Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli.

    PubMed Central

    Laviña, M; Gaggero, C; Moreno, F

    1990-01-01

    Microcin H47 (MccH47) is a novel microcin antibiotic produced by a natural Escherichia coli isolate. In contrast to all the other colicins and microcins examined to date, which are plasmid encoded, the genes for MccH47 synthesis and immunity are located on the chromosome. These genetic determinants were cloned and shown to extend over a continuous DNA region of ca. 10 kb. Images PMID:2228975

  20. FtsK actively segregates sister chromosomes in Escherichia coli.

    PubMed

    Stouf, Mathieu; Meile, Jean-Christophe; Cornet, François

    2013-07-02

    Bacteria use the replication origin-to-terminus polarity of their circular chromosomes to control DNA transactions during the cell cycle. Segregation starts by active migration of the region of origin followed by progressive movement of the rest of the chromosomes. The last steps of segregation have been studied extensively in the case of dimeric sister chromosomes and when chromosome organization is impaired by mutations. In these special cases, the divisome-associated DNA translocase FtsK is required. FtsK pumps chromosomes toward the dif chromosome dimer resolution site using polarity of the FtsK-orienting polar sequence (KOPS) DNA motifs. Assays based on monitoring dif recombination have suggested that FtsK acts only in these special cases and does not act on monomeric chromosomes. Using a two-color system to visualize pairs of chromosome loci in living cells, we show that the spatial resolution of sister loci is accurately ordered from the point of origin to the dif site. Furthermore, ordered segregation in a region ∼200 kb long surrounding dif depended on the oriented translocation activity of FtsK but not on the formation of dimers or their resolution. FtsK-mediated segregation required the MatP protein, which delays segregation of the dif-surrounding region until cell division. We conclude that FtsK segregates the terminus region of sister chromosomes whether they are monomeric or dimeric and does so in an accurate and ordered manner. Our data are consistent with a model in which FtsK acts to release the MatP-mediated cohesion and/or interaction with the division apparatus of the terminus region in a KOPS-oriented manner.

  1. Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli, Avoiding Dangers of Runaway Overreplication

    PubMed Central

    Khan, Sharik R.; Mahaseth, Tulip; Kouzminova, Elena A.; Cronan, Glen E.; Kuzminov, Andrei

    2016-01-01

    We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. PMID:26801182

  2. Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli

    PubMed Central

    Peter, Brian J; Arsuaga, Javier; Breier, Adam M; Khodursky, Arkady B; Brown, Patrick O; Cozzarelli, Nicholas R

    2004-01-01

    Background The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure. Results We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions. Conclusions The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell. PMID:15535863

  3. GENETIC RECOMBINATION BETWEEN THE RESISTANCE TRANSFER FACTOR AND THE CHROMOSOME OF ESCHERICHIA COLI

    PubMed Central

    Ginoza, Herbert S.; Painter, Robert B.

    1964-01-01

    Ginoza, Herbert S. (National Aeronautics and Space Administration, Moffett Field, Calif.), and Robert B. Painter. Genetic recombination between the resistance transfer factor and the chromosome of Escherichia coli. J. Bacteriol. 87:1339–1345. 1964.—Genetic instability for high-level streptomycin and chloramphenicol resistance was observed in several strains of Escherichia coli infected with the resistance transfer factor (RTF) episome. The altered site was always found on the chromosome, and the resistance characteristics were similar to, if not identical with, the corresponding determinant found on the episome. The high-level drug resistance phenotype was ascribed to two separate loci acting cooperatively within the host. The instability phenomenon had been attributed to a genetic exchange mechanism in which the chromosome copies the drug-resistance information from the episome, thus giving rise to a diploid homogenote for this segment. In a reciprocal exchange system, the tetracycline-resistance marker on the chromosome was shown to recombine with the RTF episome lacking this information. PMID:14188711

  4. Features of genomic organization in a nucleotide-resolution molecular model of the Escherichia coli chromosome.

    PubMed

    Hacker, William C; Li, Shuxiang; Elcock, Adrian H

    2017-07-27

    We describe structural models of the Escherichia coli chromosome in which the positions of all 4.6 million nucleotides of each DNA strand are resolved. Models consistent with two basic chromosomal orientations, differing in their positioning of the origin of replication, have been constructed. In both types of model, the chromosome is partitioned into plectoneme-abundant and plectoneme-free regions, with plectoneme lengths and branching patterns matching experimental distributions, and with spatial distributions of highly-transcribed chromosomal regions matching recent experimental measurements of the distribution of RNA polymerases. Physical analysis of the models indicates that the effective persistence length of the DNA and relative contributions of twist and writhe to the chromosome's negative supercoiling are in good correspondence with experimental estimates. The models exhibit characteristics similar to those of 'fractal globules,' and even the most genomically-distant parts of the chromosome can be physically connected, through paths combining linear diffusion and inter-segmental transfer, by an average of only ∼10 000 bp. Finally, macrodomain structures and the spatial distributions of co-expressed genes are analyzed: the latter are shown to depend strongly on the overall orientation of the chromosome. We anticipate that the models will prove useful in exploring other static and dynamic features of the bacterial chromosome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on dam methylation.

    PubMed

    Koch, Birgit; Ma, Xiaofang; Løbner-Olesen, Anders

    2010-08-01

    We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

  6. Physical manipulation of the Escherichia coli chromosome reveals its soft nature.

    PubMed

    Pelletier, James; Halvorsen, Ken; Ha, Bae-Yeun; Paparcone, Raffaella; Sandler, Steven J; Woldringh, Conrad L; Wong, Wesley P; Jun, Suckjoon

    2012-10-02

    Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique "micropiston" and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 10(5) k(B)T are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction-decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.

  7. Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-10-05

    Escherichia coli K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium. We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the E. coli chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli. In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of E. coli.

  8. Production of shikimic acid from Escherichia coli through chemically inducible chromosomal evolution and cofactor metabolic engineering

    PubMed Central

    2014-01-01

    Background Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production. Results The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG fbr , aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol. Conclusion An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was

  9. Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

    PubMed

    Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

    2016-02-01

    Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

  10. Mapping of Escherichia Coli Chromosomal Tn5 and F Insertions by Pulsed Field Gel Electrophoresis

    PubMed Central

    Smith, C. L.; Kolodner, R. D.

    1988-01-01

    A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements. PMID:2840334

  11. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  12. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  13. Speculations on the initiation of chromosome replication in Escherichia coli: the dualism hypothesis.

    PubMed

    Norris, Vic

    2011-05-01

    The exact nature of the mechanism that triggers initiation of chromosome replication in the best understood of all organisms, Escherichia coli, remains mysterious. Here, I suggest that this mechanism evolved in response to the problems that arise if chromosome replication does not occur. E. coli is now known to be highly structured. This leads me to propose a mechanism for initiation of replication based on the dynamics of large assemblies of molecules and macromolecules termed hyperstructures. In this proposal, hyperstructures and their constituents are put into two classes, non-equilibrium and equilibrium, that spontaneously separate and that are appropriate for life in either good or bad conditions. Maintaining the right ratio(s) of non-equilibrium to equilibrium hyperstructures is therefore a major challenge for cells. I propose that this maintenance entails a major transfer of material from equilibrium to non-equilibrium hyperstructures once per cell and I further propose that this transfer times the cell cycle. More specifically, I speculate that the dialogue between hyperstructures involves the structuring of water and the condensation of cations and that one of the outcomes of ion condensation on ribosomal hyperstructures and decondensation from the origin hyperstructure is the separation of strands at oriC responsible for triggering initiation of replication. The dualism hypothesis that comes out of these speculations may help integrate models for initiation of replication, chromosome segregation and cell division with the 'prebiotic ecology' scenario of the origins of life. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Kinetics of large-scale chromosomal movement during asymmetric cell division in Escherichia coli

    PubMed Central

    Männik, Jaana; O’Neill, Jordan C.

    2017-01-01

    Coordination between cell division and chromosome replication is essential for a cell to produce viable progeny. In the commonly accepted view, Escherichia coli realize this coordination via the accurate positioning of its cell division apparatus relative to the nucleoids. However, E. coli lacking proper positioning of its cell division planes can still successfully propagate. Here, we characterize how these cells partition their chromosomes into daughters during such asymmetric divisions. Using quantitative time-lapse imaging, we show that DNA translocase, FtsK, can pump as much as 80% (3.7 Mb) of the chromosome between daughters at an average rate of 1700±800 bp/s. Pauses in DNA translocation are rare, and in no occasions did we observe reversals at experimental time scales of a few minutes. The majority of DNA movement occurs at the latest stages of cell division when the cell division protein ZipA has already dissociated from the septum, and the septum has closed to a narrow channel with a diameter much smaller than the resolution limit of the microscope (~250 nm). Our data suggest that the narrow constriction is necessary for effective translocation of DNA by FtsK. PMID:28234902

  15. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome.

    PubMed

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-05

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  16. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    PubMed Central

    Sneppen, Kim; Semsey, Szabolcs

    2014-01-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome. PMID:25475788

  17. A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome.

    PubMed

    Windle, B E; Hays, J B

    1986-06-01

    Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically.

  18. A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome.

    PubMed Central

    Windle, B E; Hays, J B

    1986-01-01

    Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically. PMID:3012538

  19. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    NASA Astrophysics Data System (ADS)

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  20. The genome-scale interplay amongst xenogene silencing, stress response and chromosome architecture in Escherichia coli

    PubMed Central

    Srinivasan, Rajalakshmi; Scolari, Vittore Ferdinando; Lagomarsino, Marco Cosentino; Seshasayee, Aswin Sai Narain

    2015-01-01

    The gene expression state of exponentially growing Escherichia coli cells is manifested by high expression of essential and growth-associated genes and low levels of stress-related and horizontally acquired genes. An important player in maintaining this homeostasis is the H-NS-StpA gene silencing system. A Δhns-stpA deletion mutant results in high expression of otherwise-silent horizontally acquired genes, many located in the terminus-half of the chromosome, and an indirect downregulation of many highly expressed genes. The Δhns-stpA double mutant displays slow growth. Using laboratory evolution we address the evolutionary strategies that E. coli would adopt to redress this gene expression imbalance. We show that two global gene regulatory mutations—(i) point mutations inactivating the stress-responsive sigma factor RpoS or σ38 and (ii) an amplification of ∼40% of the chromosome centred around the origin of replication—converge in partially reversing the global gene expression imbalance caused by Δhns-stpA. Transcriptome data of these mutants further show a three-way link amongst the global gene regulatory networks of H-NS and σ38, as well as chromosome architecture. Increasing gene expression around the terminus of replication results in a decrease in the expression of genes around the origin and vice versa; this appears to be a persistent phenomenon observed as an association across ∼300 publicly-available gene expression data sets for E. coli. These global suppressor effects are transient and rapidly give way to more specific mutations, whose roles in reversing the growth defect of H-NS mutations remain to be understood. PMID:25429971

  1. The genome-scale interplay amongst xenogene silencing, stress response and chromosome architecture in Escherichia coli.

    PubMed

    Srinivasan, Rajalakshmi; Scolari, Vittore Ferdinando; Lagomarsino, Marco Cosentino; Seshasayee, Aswin Sai Narain

    2015-01-01

    The gene expression state of exponentially growing Escherichia coli cells is manifested by high expression of essential and growth-associated genes and low levels of stress-related and horizontally acquired genes. An important player in maintaining this homeostasis is the H-NS-StpA gene silencing system. A Δhns-stpA deletion mutant results in high expression of otherwise-silent horizontally acquired genes, many located in the terminus-half of the chromosome, and an indirect downregulation of many highly expressed genes. The Δhns-stpA double mutant displays slow growth. Using laboratory evolution we address the evolutionary strategies that E. coli would adopt to redress this gene expression imbalance. We show that two global gene regulatory mutations-(i) point mutations inactivating the stress-responsive sigma factor RpoS or σ38 and (ii) an amplification of ∼40% of the chromosome centred around the origin of replication-converge in partially reversing the global gene expression imbalance caused by Δhns-stpA. Transcriptome data of these mutants further show a three-way link amongst the global gene regulatory networks of H-NS and σ38, as well as chromosome architecture. Increasing gene expression around the terminus of replication results in a decrease in the expression of genes around the origin and vice versa; this appears to be a persistent phenomenon observed as an association across ∼300 publicly-available gene expression data sets for E. coli. These global suppressor effects are transient and rapidly give way to more specific mutations, whose roles in reversing the growth defect of H-NS mutations remain to be understood.

  2. Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

    ERIC Educational Resources Information Center

    Rotman, Ella Rose

    2009-01-01

    Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

  3. Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

    ERIC Educational Resources Information Center

    Rotman, Ella Rose

    2009-01-01

    Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

  4. Use of gene fusions to determine the orientation of gene phoA on the Escherichia coli chromosome.

    PubMed Central

    Sarthy, A; Michaelis, S; Beckwith, J

    1981-01-01

    We present genetic evidence which demonstrates that the phoA gene is transcribed in the clockwise direction on the Escherichia coli chromosome, in contrast to an earlier proposal. Our conclusion is based on analysis of various genetic fusions between the lac operon and the phoA gene. PMID:7007316

  5. Membrane attachment activates dnaA protein, the initiation protein of chromosome replication in Escherichia coli

    SciTech Connect

    Yung, B.Y.; Kornberg, A.

    1988-10-01

    ADP and ATP are tightly bound to dnaA protein and are crucial to its function in DNA replication; the exchange of these nucleotides is effected specifically by the acidic phospholipids (cardiolipin and phosphatidylglycerol) present in Escherichia coli membranes. We now find that phospholipids derived from membranes lacking an unsaturated fatty acid (e.g., oleic acid) are unable to promote the exchange. This observation correlates strikingly with the long-known effect of 3-decynoyl-N-acetylcysteamine, a ''suicide analog'' that prevents initiation of a cycle of replication in E. coli by inhibiting the synthesis of oleic acid, an inhibition that can be overcome by providing the cells with oleic acid. Profound influences on the specific binding of dnaA protein to phospholipids by temperature, the content of unsaturated fatty acids, and the inclusion of cholesterol can be explained by the need for the phospholipids to be in fluid-phase vesicles. These findings suggest that membrane attachment of dnaA protein is vital for its function in the initiation of chromosome replication in E. coli.

  6. Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

    PubMed Central

    Tresguerres, E F; Nieto, C; Casquero, I; Cánovas, J L

    1986-01-01

    Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell. PMID:3511032

  7. Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

    PubMed Central

    Aedo, Sandra

    2012-01-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA. PMID:22869559

  8. Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA.

    PubMed

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-11-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

  9. Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli.

    PubMed

    Raynal, A; Tuphile, K; Gerbaud, C; Luther, T; Guérineau, M; Pernodet, J L

    1998-04-01

    The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

  10. Many chromosomal genes modulate MarA-mediated multidrug resistance in Escherichia coli.

    PubMed

    Ruiz, Cristian; Levy, Stuart B

    2010-05-01

    Multidrug resistance (MDR) in clinical isolates of Escherichia coli can be associated with overexpression of marA, a transcription factor that upregulates multidrug efflux and downregulates membrane permeability. Using random transposome mutagenesis, we found that many chromosomal genes and environmental stimuli affected MarA-mediated antibiotic resistance. Seven genes affected resistance mediated by MarA in an antibiotic-specific way; these were mostly genes encoding unrelated enzymes, transporters, and unknown proteins. Other genes affected MarA-mediated resistance to all antibiotics tested. These genes were acrA, acrB, and tolC (which encode the major MarA-regulated multidrug efflux pump AcrAB-TolC), crp, cyaA, hns, and pcnB (four genes involved in global regulation of gene expression), and the unknown gene damX. The last five genes affected MarA-mediated MDR by altering marA expression or MarA function specifically on acrA. These findings demonstrate that MarA-mediated MDR is regulated at multiple levels by different genes and stimuli, which makes it both complex and fine-tuned and interconnects it with global cell regulation and metabolism. Such a regulation could contribute to the adaptation and spread of MDR strains and may be targeted to treat antibiotic-resistant E. coli and related pathogens.

  11. Clonal expansion of Escherichia coli ST38 carrying a chromosomally integrated OXA-48 carbapenemase gene.

    PubMed

    Turton, Jane F; Doumith, Michel; Hopkins, Katie L; Perry, Claire; Meunier, Daniele; Woodford, Neil

    2016-06-01

    Many isolates of Escherichia coli carrying blaOXA-48 referred to Public Health England's national reference laboratory during 2014 and 2015 shared similar pulsed-field gel electrophoresis (PFGE) profiles, despite coming from patients in multiple different hospitals and regions. Whole genome sequencing on an Illumina platform revealed that these belonged to sequence type (ST) 38. The OXA-48 gene is usually carried on a 62 kb IncL/M plasmid (pOXA48a), but those belonging to this ST appeared either to lack plasmid elements or to have only a partial complement. Two isolates, one belonging to a main cluster sharing identical PFGE profiles and the other having a distinct profile, were further sequenced on a minION. The long reads provided by the nanopore sequencing technology facilitated assembly of a much larger contig around the blaOXA-48 region, showing that both isolates shared a similar arrangement, with a plasmid fragment containing blaOXA-48 flanked by IS1R elements integrated into the chromosome, although the length of the plasmid fragment and the insertion site differed between the two isolates. That belonging to the main cluster contained a 21.9 kb Tn6237 insert, as previously described in E. coli EC-15 from Lebanon, but in a different insertion site. PCR mapping indicated that a further 14/31 representatives of this cluster also contained this insert in the same insertion site, with most of the remainder differing only by having additional E. coli sequence on one side of the insertion. This sub-cluster of ST38 was found from 25 different hospital laboratories, suggesting widespread distribution of a successful type.

  12. Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

    PubMed

    Itaya, M

    1995-07-22

    A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

  13. Bacteriophage P1 pac sites inserted into the chromosome greatly increase packaging and transduction of Escherichia coli genomic DNA.

    PubMed

    Huang, Haomin; Masters, Millicent

    2014-11-01

    The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.

  14. Scanning the Escherichia coli chromosome by random transposon mutagenesis and multiple phenotypic screening.

    PubMed

    Serina, Stefania; Nozza, Francesca; Nicastro, Giovanna; Faggioni, Federico; Mottl, Harald; Dehò, Gianni; Polissi, Alessandra

    2004-10-01

    Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araP(BAD) promoter oriented outward at one end (Tn5-araP(BAD)). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15 degrees C) and in different media (rich and minimal medium). The Tn5-araP(BAD)-tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37 degrees C) but not in the cold and in minimal medium.

  15. [Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

    PubMed

    Li, Shanhu; Shi, Qingguo; Huang, Cuifen; Zhou, Jianguang

    2008-04-01

    To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

  16. Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions

    PubMed Central

    Poteete, Anthony R

    2009-01-01

    Background Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied. Results Under selection for lac function, colonies bearing multiple copies of the mutant lac operon appeared at a constant rate of approximately 4 to 5 per million cells plated per day, on days two through seven after plating. Expansion was not seen in a recA strain; null mutations in recBCD and ruvC reduced the rate 100- and 10-fold, respectively; a ruvC recG double mutant reduced the rate 1000-fold. Expansion occurred at an increased rate in cells lacking dam, polA, rnhA, or uvrD functions. Null mutations of various other cellular recombination, repair, and stress response genes had little effect upon expansion. The red recombination genes of phage lambda could substitute for recBCD in mediating expansion. In the red-substituted cells, expansion was only partially dependent upon recA function. Conclusion These observations are consistent with the idea that the expansion step of gene amplification is closely related, mechanistically, to interchromosomal homologous recombination events. They additionally provide support for recently described models of RecA-independent Red-mediated recombination at replication forks. PMID:19236706

  17. RNase HII Saves rnhA Mutant Escherichia coli from R-Loop-Associated Chromosomal Fragmentation.

    PubMed

    Kouzminova, Elena A; Kadyrov, Farid F; Kuzminov, Andrei

    2017-09-15

    The rnhAB mutant Escherichia coli, deficient in two RNase H enzymes that remove both R-loops and incorporated ribonucleotides (rNs) from DNA, grow slowly, suggesting accumulation of rN-containing DNA lesions (R-lesions). We report that the rnhAB mutants have reduced viability, form filaments with abnormal nucleoids, induce SOS, and fragment their chromosome, revealing replication and/or segregation stress. R-loops are known to interfere with replication forks, and sensitivity of the double rnhAB mutants to translation inhibition points to R-loops as precursors for R-lesions. However, the strict specificity of bacterial RNase HII for RNA-DNA junctions indicates that R-lesions have rNs integrated into DNA. Indeed, instead of relieving problems of rnhAB mutants, transient inhibition of replication from oriC kills them, suggesting that oriC-initiated replication removes R-loops instead of compounding them to R-lesions. Yet, replication from an R-loop-initiating plasmid origin kills the double rnhAB mutant, revealing generation of R-lesions by R-loop-primed DNA synthesis. These R-lesions could be R-tracts, contiguous runs of ≥4 RNA nucleotides within DNA strand and the only common substrate between the two bacterial RNase H enzymes. However, a plasmid relaxation test failed to detect R-tracts in DNA of the rnhAB mutants, although it readily detected R-patches (runs of 1-3 rNs). Instead, we detected R-gaps, single-strand gaps containing rNs, in the chromosomal DNA of the rnhAB mutant. Therefore, we propose that RNase H-deficient mutants convert some R-loops into R-tracts, which progress into R-gaps and then to double-strand breaks-explaining why R-tracts do not accumulate in RNase H-deficient cells, while double-strand breaks do. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome.

    PubMed

    Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

    2012-01-01

    We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF (fbr) and tyrA (fbr) or aroF (fbr) and pheA (fbr)) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

  19. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  20. Computational analyses of transcriptomic data reveal the dynamic organization of the Escherichia coli chromosome under different conditions

    PubMed Central

    Ma, Qin; Yin, Yanbin; Schell, Mark A.; Zhang, Han; Li, Guojun; Xu, Ying

    2013-01-01

    The circular chromosome of Escherichia coli has been suggested to fold into a collection of sequentially consecutive domains, genes in each of which tend to be co-expressed. It has also been suggested that such domains, forming a partition of the genome, are dynamic with respect to the physiological conditions. However, little is known about which DNA segments of the E. coli genome form these domains and what determines the boundaries of these domain segments. We present a computational model here to partition the circular genome into consecutive segments, theoretically suggestive of the physically folded supercoiled domains, along with a method for predicting such domains under specified conditions. Our model is based on a hypothesis that the genome of E. coli is partitioned into a set of folding domains so that the total number of unfoldings of these domains in the folded chromosome is minimized, where a domain is unfolded when a biological pathway, consisting of genes encoded in this DNA segment, is being activated transcriptionally. Based on this hypothesis, we have predicted seven distinct sets of such domains along the E. coli genome for seven physiological conditions, namely exponential growth, stationary growth, anaerobiosis, heat shock, oxidative stress, nitrogen limitation and SOS responses. These predicted folding domains are highly stable statistically and are generally consistent with the experimental data of DNA binding sites of the nucleoid-associated proteins that assist the folding of these domains, as well as genome-scale protein occupancy profiles, hence supporting our proposed model. Our study established for the first time a strong link between a folded E. coli chromosomal structure and the encoded biological pathways and their activation frequencies. PMID:23599001

  1. Cloning and deletion mapping of the recF dnaN region of the Escherichia coli chromosome.

    PubMed

    Ream, L W; Clark, A J

    1983-09-01

    By cloning a 3.6-kb EcoRI fragment of the Escherichia coli chromosome with pBR322 we located more precisely recF relative to dnaN. By deletion mapping we localized functional recF to a 1.65-kb region of the cloned fragment and allowed rough mapping of the C terminus of dnaN. Cloned recF+, separated from functional flanking genes dnaN and gyrB, complemented chromosomal recF mutations presumably by coding for a cytodiffusible product. The protein encoded by dnaN was observed as a band on a polyacrylamide gel from minicells. Identification of a recF protein was not made.

  2. Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain.

    PubMed Central

    Hacker, J; Knapp, S; Goebel, W

    1983-01-01

    The hemolytic Escherichia coli strain 536 (O6) propagates spontaneous hemolysin-negative mutants at relatively high rates (10(-3) to 10(-4)). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (Hlyex-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ and Mrh+. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type II lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants. Images PMID:6343344

  3. Crosstalk between DnaA Protein, the Initiator of Escherichia coli Chromosomal Replication, and Acidic Phospholipids Present in Bacterial Membranes

    PubMed Central

    Saxena, Rahul; Fingland, Nicholas; Patil, Digvijay; Sharma, Anjali K.; Crooke, Elliott

    2013-01-01

    Anionic (i.e., acidic) phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL), participate in several cellular functions. Here we review intriguing in vitro and in vivo evidence that suggest emergent roles for acidic phospholipids in regulating DnaA protein-mediated initiation of Escherichia coli chromosomal replication. In vitro acidic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA, yet both PG and CL also can inhibit the DNA-binding activity of DnaA protein. We discuss how cellular acidic phospholipids may positively and negatively influence the initiation activity of DnaA protein to help assure chromosomal replication occurs once, but only once, per cell-cycle. Fluorescence microscopy has revealed that PG and CL exist in domains located at the cell poles and mid-cell, and several studies link membrane curvature with sub-cellular localization of various integral and peripheral membrane proteins. E. coli DnaA itself is found at the cell membrane and forms helical structures along the longitudinal axis of the cell. We propose that there is cross-talk between acidic phospholipids in the bacterial membrane and DnaA protein as a means to help control the spatial and temporal regulation of chromosomal replication in bacteria. PMID:23595001

  4. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.

    PubMed

    Diorio, C; Cai, J; Marmor, J; Shinder, R; DuBow, M S

    1995-04-01

    Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.

  5. Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

    PubMed Central

    Dubarry, Nelly; Barre, François-Xavier

    2010-01-01

    In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum. PMID:20033058

  6. Interplay between plasmid-mediated and chromosomal-mediated fluoroquinolone resistance and bacterial fitness in Escherichia coli.

    PubMed

    Machuca, Jesús; Briales, Alejandra; Labrador, Gema; Díaz-de-Alba, Paula; López-Rojas, Rafael; Docobo-Pérez, Fernando; Martínez-Martínez, Luis; Rodríguez-Baño, Jesús; Pachón, Maria Eugenia; Pascual, Alvaro; Rodríguez-Martínez, José-Manuel

    2014-12-01

    The aim of this study was to analyse the interplay among plasmid-mediated qnr genes, alone or in combination with multiple chromosomal-mediated fluoroquinolone (FQ) resistance determinants, susceptibility to FQs and bacterial fitness in an isogenic Escherichia coli collection. E. coli ATCC 25922 was used to modify or delete chromosomal genes. qnr genes were cloned into the pBK-CMV vector. The MICs of FQs were determined by microdilution. Mutant prevention concentration and frequency of mutants were evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays using in vitro and in vivo models. The relationships between the number of resistance mutations and bacterial fitness were complex. With specific combinations of resistance mechanisms the addition of a new resistance mutation was shown to improve bacterial fitness. qnrA1 caused a decrease in fitness (7%-21%) while qnrS1 caused an increase in fitness (9%-21%) when combined with chromosomal mutations. We identified susceptible triple mutants in which the acquisition of a fourth resistance mutation significantly increased fitness and at the same time reached the clinical resistance level (the acquisition of qnrS1 in a S83L + D87N + ΔmarR genetic background). A strong correlation with the production of reactive oxygen species, as well as changes in susceptibility, was observed following treatment with ciprofloxacin. Our data indicate that there may be critical stages (depending on the genotype) in resistance development, including chromosomal- and plasmid-mediated mechanisms, at which some low-fitness mutants below the resistance breakpoint are able to evolve clinical resistance with just one or two mutations, and show increased fitness. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. SOE-LRed: a simple and time-efficient method to localize genes with point mutations onto the Escherichia coli chromosome

    PubMed Central

    Benson, Ryan W.; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2011-01-01

    We report a powerful method to replace wild type genes on the chromosome of Escherichia coli. Employing a unique form of PCR, we generate easily constructible gene fusions bearing single point mutations. Used in conjunction with homologous recombination, this method eliminates cloning procedures previously used for this purpose. PMID:21185880

  8. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo

    PubMed Central

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L.; Stavans, Joel

    2014-01-01

    The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host. PMID:24799672

  9. Proteomic identification of the Cus system as a major determinant of constitutive Escherichia coli silver resistance of chromosomal origin.

    PubMed

    Lok, Chun-Nam; Ho, Chi-Ming; Chen, Rong; Tam, Paul Kwong-Hang; Chiu, Jen-Fu; Che, Chi-Ming

    2008-06-01

    Although silver is one of the most potent and rapidly acting toxic metals to bacteria, silver-resistant bacteria do exist with low incidence. A proteomic approach was employed to identify the silver resistance determinants of a silver-resistant Escherichia coli strain isolated from stepwise selection against increasing concentrations of silver (Li et al. J. Bacteriol 1997, 179, 6127-32). Two-dimensional gel electrophoresis and mass spectrometry analysis revealed that members of the CusCFBA copper/silver chemiosmotic efflux system were highly expressed in the silver-resistant strain but undetectable in the parental silver-sensitive strain. Disruption of the cus locus of the silver-resistant strain resulted in a decrease of the minimum inhibitory concentration of Ag (+) from more than 1 mM to 12 microM. These results suggest that the chromosomally encoded Cus system, which naturally controls the periplasmic copper concentrations, is selectable to confer a constitutive silver resistance phenotype.

  10. A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands.

    PubMed

    Azeroglu, Benura; Lincker, Frédéric; White, Martin A; Jain, Devanshi; Leach, David R F

    2014-12-01

    DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination.

  11. A standard vector for the chromosomal integration and characterization of BioBrick™ parts in Escherichia coli

    PubMed Central

    2013-01-01

    Background The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context. Results In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick™ standards in Escherichia coli. It can also be specialized by using BioBrick™ parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick™ devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions. Conclusions Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick™ vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick™ parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use. PMID:23663425

  12. Efficient chromosomal-scale DNA looping in Escherichia coli using multiple DNA-looping elements.

    PubMed

    Hao, Nan; Sneppen, Kim; Shearwin, Keith E; Dodd, Ian B

    2017-05-19

    Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. But it is not understood how efficient looping can occur when the sites are very far apart. We develop a simple theoretical framework that relates looping efficiency to the energetic cost and benefit of looping, allowing prediction of the efficiency of single or multiple nested loops at different distances. Measurements of absolute loop efficiencies for Lac repressor and λ CI using gene expression reporters in Escherichia coli cells show that, as predicted by the model, long-range DNA looping between a pair of sites can be strongly enhanced by the use of nested DNA loops or by the use of additional protein-binding sequences. A combination of these approaches was able to generate efficient DNA looping at a 200 kb distance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. in vivo Measurements of Conformational Fluctuations of Chromosomal DNA in Escherichia Coli

    NASA Astrophysics Data System (ADS)

    Kafle, Rudra; Meiners, Jens-Christian

    2013-03-01

    The cell is the site of active, motor-driven processes far from thermodynamic equilibrium. Therefore, the intracellular dynamics are complex and subject to a multitude of constraints and forces. We study the conformational fluctuations of chromosomal DNA in vivo in live and dead E. coli cells by Fluorescence Correlation Spectroscopy (FCS). The fluctuations move the DNA-bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a confocal microscope. From the time correlation functions of the fluorescence intensity, we obtain the mean square displacements of the DNA on a time scale from microseconds to seconds. We see a substantial decrease in the power spectral density (PSD) of the displacement fluctuations at frequencies below 10 Hz in the dead cells, compared to the live cells. The larger fluctuations in the living cells may indicate that the fluctuations on this time scale may be driven by active processes involving molecular motors that generate forces by ATP hydrolysis. A small difference in PSD between live and dead cells on shorter time scales suggests that the processes on corresponding short length scales rely primarily on thermally-driven diffusive mechanisms.

  14. Association of Mu-containing plasmids with the Escherichia coli chromosome upon prophage induction

    PubMed Central

    Chaconas, George; Harshey, Rasika M.; Bukhari, Ahmad I.

    1980-01-01

    To determine the structure of a prophage-containing plasmid during Mu transposition, we have monitored the physical state of pSC101[unk]Mucts after thermoinduction. We have also examined the fate of a mini Mu plasmid constructed in vitro by deleting 27 kilobases from the center of the Mu prophage in pSC101[unk]Mucts. At various times after prophage induction, DNA was extracted from Mu or mini Mu plasmid-containing strains and subjected to electrophoresis in low concentration agarose gels followed by transfer of the DNA to nitrocellulose paper. Separate hybridization with 32P-labeled pSC101 and Mu DNA revealed the position of the plasmids and the replication of Mu DNA. At times after induction when Mu replication was clearly visible, Mu and mini Mu plasmids were found to migrate with Escherchia coli DNA. This Mu-specific association requires the phage coded A and B proteins. Electron microscopy has shown that some of the associated DNA is comprised of circular plasmid molecules which appear to be in contact with the chromosomal DNA. These structures may represent intermediates or end products of the replication-integration process. The finding that Mu and mini Mu plasmids do not give rise to any detectable excision products and apparently remain intact during Mu transposition supports our proposal that the predominant event after Mu induction is the replication of Mu DNA in situ to generate integrative intermediates. Images PMID:6246503

  15. The chromosomal toxin gene yafQ is a determinant of multidrug tolerance for Escherichia coli growing in a biofilm.

    PubMed

    Harrison, Joe J; Wade, William D; Akierman, Sarah; Vacchi-Suzzi, Caterina; Stremick, Carol A; Turner, Raymond J; Ceri, Howard

    2009-06-01

    Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a Delta yafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the Delta yafQ strain had up to a approximately 2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a approximately 10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.

  16. The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm▿

    PubMed Central

    Harrison, Joe J.; Wade, William D.; Akierman, Sarah; Vacchi-Suzzi, Caterina; Stremick, Carol A.; Turner, Raymond J.; Ceri, Howard

    2009-01-01

    Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a ΔyafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the ΔyafQ strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures. PMID:19307375

  17. Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

    PubMed

    Akiyama, Masahiro Tatsumi; Oshima, Taku; Chumsakul, Onuma; Ishikawa, Shu; Maki, Hisaji

    2016-08-01

    Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

  18. Localized remodeling of the Escherichia coli chromosome: the patchwork of segments refractory and tolerant to inversion near the replication terminus.

    PubMed Central

    Guijo, M I; Patte, J; del Mar Campos, M; Louarn, J M; Rebollo, J E

    2001-01-01

    The behavior of chromosomal inversions in Escherichia coli depends upon the region they affect. Regions flanking the replication terminus have been termed nondivisible zones (NDZ) because inversions ending in the region were either deleterious or not feasible. This regional phenomenon is further analyzed here. Thirty segments distributed between 23 and 29 min on the chromosome map have been submitted to an inversion test. Twenty-five segments either became deleterious when inverted or were noninvertible, but five segments tolerated inversion. The involvement of polar replication pause sites in this distribution was investigated. The results suggest that the Tus/pause site system may forbid some inversion events, but that other constraints to inversion, unrelated to this system, exist. Our current model for deleterious inversions is that the segments involved carry polar sequences acting in concert with other polar sequences located outside the segments. The observed patchwork of refractory and tolerant segments supports the existence of several NDZs in the 23- to 29-min region. Microscopic observations revealed that deleterious inversions are associated with high frequencies of abnormal nucleoid structure and distribution. Combined with other information, the data suggest that NDZs participate in the organization of the terminal domain of the nucleoid. PMID:11290700

  19. Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132.

    PubMed

    Bockmann, J; Heuel, H; Lengeler, J W

    1992-10-01

    A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E. coli K12. EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype. The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB. Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids. CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type. A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.

  20. Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome

    SciTech Connect

    Takase, I.; Ishino, F.; Wachi, M.; Kamata, H.; Doi, M.; Asoh, S.; Matsuzawa, H.; Ohta, T.; Matsuhashi, M.

    1987-12-01

    The coding of two rare lipoproteins by two genes, rlpA and rlpB, located in the leuS-dacA region (15 min) on the Escherichia coli chromosome was demonstrated by expression of subcloned genes in a maxicell system. The formation of these two proteins was inhibited by globomycin, which is an inhibitor of the signal peptidase for the known lipoproteins of E. coli. In each case, this inhibition was accompanied by formation of a new protein, which showed a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which we suppose to be a prolipoprotein with an N-terminal signal peptide sequence similar to those of the bacterial major lipoproteins and lysis proteins of some bacteriocins. The incorporation of /sup 3/H-labeled palmitate and glycerol into the two lipoproteins was also observed. Sequencing of DNA showed that the two lipoprotein genes contained sequences that could code for signal peptide sequences of 17 amino acids (rlpA lipoprotein) and 18 amino acids (rlpB lipoprotein). The deduced sequences of the mature peptides consisted of 345 amino acids (M/sub r/ 35,615, rlpA lipoprotein) and 175 amino acids (M/sub r/ 19,445, rlpB lipoprotein), with an N-terminal cysteine to which thioglyceride and N-fatty acyl residues may be attached. These two lioproteins may be important in duplication of the cells.

  1. IS1R-mediated plasticity of IncL/M plasmids leads to the insertion of bla OXA-48 into the Escherichia coli Chromosome.

    PubMed

    Beyrouthy, R; Robin, F; Delmas, J; Gibold, L; Dalmasso, G; Dabboussi, F; Hamzé, M; Bonnet, R

    2014-07-01

    The OXA-48 carbapenemase is mainly encoded by ∼ 62-kb IncL/M plasmids. However, chromosome-mediated genes have been observed in Escherichia coli isolates. In this work, we investigated the genetic environment of OXA-48 in members of the family Enterobacteriaceae (n = 22) to understand how the OXA-48-encoding gene is transferred into the E. coli chromosome. The OXA-48-encoding gene was located within intact Tn1999.2 transposons in the ∼ 62-kb plasmids or within a truncated variant of Tn1999.2 for the OXA-48-encoding genes located in the chromosomes of E. coli bacteria. The analysis of the Tn1999.2 genetic environment revealed an inverted orientation of the transposon in five ∼ 62-kb plasmids (5/14 [35%]) and in all chromosome inserts (n = 8). The sequencing of pRA35 plasmid showed that this orientation of Tn1999.2 and the acquisition of an IS1R insertion sequence generated a 21.9-kb IS1R-based composite transposon encoding OXA-48 and designated Tn6237. The sequencing of a chromosomal insert encoding OXA-48 also revealed this new transposon in the E. coli chromosome. PCR mapping showed the presence of this element in all strains harboring an OXA-48-encoding chromosomal insert. However, different insertion sites of this transposon were observed in the E. coli chromosome. Overall, these findings indicate a plasticity of the OXA-48 genetic environment mediated by IS1R insertion sequences. The insertion sequences can induce the transfer of the OXA-encoding gene into E. coli chromosomes and thereby promote its persistence and expression at low levels.

  2. Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

    PubMed Central

    2009-01-01

    Background Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Results Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. Conclusion This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones. PMID:19364397

  3. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  4. Overproduction of Three Genes Leads to Camphor Resistance and Chromosome Condensation in Escherichia Coli

    PubMed Central

    Hu, K. H.; Liu, E.; Dean, K.; Gingras, M.; DeGraff, W.; Trun, N. J.

    1996-01-01

    We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB. Both phenotypes require the same genes. Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells. The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested. We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense. Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding. PMID:8844142

  5. Pathogenic Escherichia coli

    USDA-ARS?s Scientific Manuscript database

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  6. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  7. Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains

    PubMed Central

    2017-01-01

    The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell. PMID:28486476

  8. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.

  9. Unique clustering genes in the bacterial chromosome affecting the type-III secretion of enterohaemorrhagic Escherichia coli.

    PubMed

    Lin, I-Ting; Chiou, Yi-Ming; Liang, Yen-Chia; Lin, Ching-Nan; Sun, Wei-Sheng W; Li, Shiaowen; Chang, Chuan-Hsiung; Syu, Wan-Jr; Chen, Jenn-Wei

    2016-10-01

    Bioinformatics analysis was used to search for unknown genes that might influence the phenotypic presentations of enterohaemorrhagic Escherichia coli (EHEC). By so doing and using the known genomic data from EHEC O157  : H7 and K-12, it has been deduced that genes Z4863 to Z4866 of EHEC do not exist in K-12 strains. These four gene sequences have low degrees of homology (18-40 % amino acid identities) to a set of genes in K-12, which have been known to encode fatty acid biosynthesis enzymes. We referred these four consecutive genes as a fasyn cluster and found that deletion of fasyn from EHEC resulted in a defective type-III secretion (T3S). This deletion apparently did not decrease the amounts of the T3S proteins ectopically expressed from plasmids. Examination of the corresponding mRNAs by real-time PCR revealed that the mRNAs readily decreased in the fasyn-deleted mutant and this suppressive effect on the mRNA levels appeared to spread across all lee operons. Complementation with fasyn reverted the T3S-deficient phenotype. Furthermore, this reversion was also seen when the mutant was supplemented with locus of enterocyte effacement activators (Ler or GrlA). Thus, these unique clustering genes located apart from locus of enterocyte effacement on the bacterial chromosome also play a role in affecting T3S of EHEC.

  10. Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

    PubMed

    Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

    2014-01-01

    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.

  11. Replication of the Escherichia coli chromosome in RNase HI-deficient cells: Multiple initiation regions and fork dynamics

    PubMed Central

    Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

    2013-01-01

    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. PMID:24164596

  12. Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2′-fucosyllactose

    PubMed Central

    2013-01-01

    Background The trisaccharide 2′-fucosyllactose (2′-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2′-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2′-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2′-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2′-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2′-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. Results Here we report the construction of the first selection marker-free E. coli strain that produces 2′-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2′-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2′-FL. Using an improved production strain, feasibility of large scale 2′-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2′-FL concentration of 20.28

  13. Escherichia coli biofilms

    PubMed Central

    Beloin, Christophe; Roux, Agnès; Ghigo, Jean-Marc

    2008-01-01

    Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli. PMID:18453280

  14. Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

    PubMed

    Gibello, Alicia; Díaz de Alba, Paula; Blanco, M Mar; Machuca, Jesus; Cutuli, M Teresa; Rodríguez-Martínez, José Manuel

    2014-09-01

    This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

  15. Diarrheagenic Escherichia coli.

    PubMed

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  16. The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

    PubMed Central

    Männik, Jaana; Castillo, Daniel E.; Yang, Da; Siopsis, George; Männik, Jaan

    2016-01-01

    Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. However, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication. PMID:26762981

  17. The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

    SciTech Connect

    Mannik, Jaana; Castillo, Daniel E.; Yang, Da; Siopsis, George; Mannik, Jaan

    2016-01-13

    Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.

  18. The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

    DOE PAGES

    Mannik, Jaana; Castillo, Daniel E.; Yang, Da; ...

    2016-01-13

    Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

  19. Chromosomal Location of the C Gene Involved in the Biosynthesis of Nicotinamide Adenine Dinucleotide in Escherichia coli K-12

    PubMed Central

    Tritz, Gerald J.; Matney, Thomas S.; Chandler, J. L. R.; Gholson, R. K.

    1970-01-01

    A gene involved in the synthesis of nicotinamide adenine dinucleotide has been found to be cotransducible with the genes involved in the utilization of arabinose (ara) and the biosynthesis of leucine (leu) and pantothenate (pan). Cotransduction frequency analysis places this nadC locus between leu and pan at approximately minute 1.5 on the genetic map of Escherichia coli. This gene codes for the enzyme, quinolate phosphoribosyl transferase, which catalyzes the conversion of quinolinic acid to nicotinic acid mononucleotide. PMID:4319723

  20. Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens.

    PubMed

    Drummelsmith, J; Amor, P A; Whitfield, C

    1997-05-01

    Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.

  1. SecA defects are accompanied by dysregulation of MukB, DNA gyrase, chromosome partitioning and DNA superhelicity in Escherichia coli.

    PubMed

    Adachi, Shun; Murakawa, Yasuhiro; Hiraga, Sota

    2014-08-01

    Spatial regulation of nucleoids and chromosome-partitioning proteins is important for proper chromosome partitioning in Escherichia coli. However, the underlying molecular mechanisms are unknown. In the present work, we showed that mutation or chemical perturbation of secretory A (SecA), an ATPase component of the membrane protein translocation machinery, SecY, a component of the membrane protein translocation channel and acyl carrier protein P (AcpP), which binds to SecA and MukB, a functional homologue of structural maintenance of chromosomes protein (SMC), resulted in a defect in chromosome partitioning. We further showed that SecA is essential for proper positioning of the oriC DNA region, decatenation and maintenance of superhelicity of DNA. Genetic interaction studies revealed that the topological abnormality observed in the secA mutant was due to combined inhibitory effects of defects in MukB, DNA gyrase and Topo IV, suggesting a role for the membrane protein translocation machinery in chromosome partitioning and/or structural maintenance of chromosomes. © 2014 The Authors.

  2. DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat

    PubMed Central

    Blackwood, John K.; Okely, Ewa A.; Zahra, Rabaab; Eykelenboom, John K.; Leach, David R. F.

    2010-01-01

    Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR. PMID:21149728

  3. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

    PubMed Central

    Juhas, Mario; Ajioka, James W

    2015-01-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. PMID:26074421

  4. An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria.

    PubMed

    Asai, T; Zaporojets, D; Squires, C; Squires, C L

    1999-03-02

    Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

  5. A Module Located at a Chromosomal Integration Hot Spot Is Responsible for the Multidrug Resistance of a Reference Strain from Escherichia coli Clonal Group A▿ †

    PubMed Central

    Lescat, Mathilde; Calteau, Alexandra; Hoede, Claire; Barbe, Valérie; Touchon, Marie; Rocha, Eduardo; Tenaillon, Olivier; Médigue, Claudine; Johnson, James R.; Denamur, Erick

    2009-01-01

    Escherichia coli clonal group A (CGA) commonly exhibits a distinctive multidrug antimicrobial resistance phenotype—i.e., resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim (ACSSuTTp)—and has accounted for up to 50% of trimethoprim-sulfamethoxazole-resistant E. coli urinary tract infections in some locales. Annotation of the whole-genome sequencing of UMN026, a reference CGA strain, clarified the genetic basis for this strain's ACSSuTTp antimicrobial resistance phenotype. Most of the responsible genes were clustered in a unique 23-kbp chromosomal region, designated the genomic resistance module (GRM), which occurred within a 105-kbp genomic island situated at the leuX tRNA. The GRM is characterized by numerous remnants of mobilization and rearrangement events suggesting multiple horizontal transfers. Additionally, comparative genomic analysis of the leuX tRNA genomic island in 14 sequenced E. coli genomes showed that this region is a hot spot of integration, with the presence/absence of specific subregions being uncorrelated with either the phylogenetic group or the pathotype. Our data illustrate the importance of whole-genome sequencing in the detection of genetic elements involved in antimicrobial resistance. Additionally, this is the first documentation of the blaTEM and dhfrVII genes in a chromosomal location in E. coli strains. PMID:19364861

  6. Site-directed mutagenesis of the Escherichia coli chromosome near oriC: identification and characterization of asnC, a regulatory element in E. coli asparagine metabolism.

    PubMed Central

    de Wind, N; de Jong, M; Meijer, M; Stuitje, A R

    1985-01-01

    We developed a new method for the specific mutagenization of the E. coli chromosome. This method takes advantage of the fact that a pBR322 plasmid containing chromosomal sequences is mobilizable during an Hfr-mediated conjugational transfer, due to an homologous recombination between the E. coli Hfr chromosome and the pBR322 derivative. Transconjugants are screened with a simple selection procedure for integration of mutant sequences in the chromosome and loss of pBR322 sequences. Using this method we specifically inactivated several genes near the E. coli replication origin oriC. We found that a gene coding for asparagine synthetase A. This regulatory mechanism was investigated in detail by determining in vivo regulation of asnA promoter activity by the 17kD protein under different growth conditions. Results obtained also suggest a general regulatory role of the 17kD protein in E. coli asparagine metabolism. Therefore the 17kD gene is proposed to be renamed asnC. Images PMID:3909107

  7. Recurrent Escherichia coli bacteremia.

    PubMed Central

    Maslow, J N; Mulligan, M E; Arbeit, R D

    1994-01-01

    Escherichia coli is the most common gram-negative organism associated with bacteremia. While recurrent E. coli urinary tract infections are well-described, recurrent E. coli bacteremia appears to be uncommon, with no episodes noted in multiple series of patients with gram-negative bacteremias. We report on 5 patients with recurrent bloodstream infections identified from a series of 163 patients with E. coli bacteremia. For each patient, the isolates from each episode were analyzed by pulsed-field gel electrophoresis (PFGE) and ribotyping and for the presence of E. coli virulence factors. For each of four patients, the index and recurrent episodes of bacteremia represented the same strain as defined by PFGE, and the strains were found to carry one or more virulence factors. The remaining patient, with two episodes of bloodstream infection separated by a 4-year interval, was infected with two isolates that did not carry any virulence factors and that were clonally related by ribotype analysis but differed by PFGE. All five patients had either a local host defense defect (three patients) or impaired systemic defenses (one patient) or both (one patient). Thus, recurrent E. coli bacteremia is likely to represent a multifactorial process that occurs in patients with impaired host defenses who are infected with virulent isolates. Images PMID:7910828

  8. ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli.

    PubMed

    Charaka, Vijaya Kumar; Mehta, Kruti P; Misra, H S

    2013-09-01

    Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the 'Min' proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.

  9. Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution. An orientation-dependent cis-effect lending support to bipolarization of the terminus.

    PubMed Central

    Corre, J; Patte, J; Louarn, J M

    2000-01-01

    A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif. In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion. TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif. We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity. By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells. The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA. Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements. Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA. PMID:10628967

  10. Enterotoxigenic Escherichia coli

    PubMed Central

    Fleckenstein, James M; Munson, George M; Rasko, David A

    2013-01-01

    The enterotoxigenic Escherichia coli are a pervasive cause of serious diarrheal illness in developing countries. Presently, there is no vaccine to prevent these infections, and many features of the basic pathogenesis of these organisms remain poorly understood. Until very recently most pathogenesis studies had focused almost exclusively on a small subset of known “classical” virulence genes, namely fimbrial colonization factors and the heat-labile (LT) and heat stable (ST) enterotoxins. However, recent investigations of pathogen-host interactions reveal a surprisingly complex and intricately orchestrated engagement involving the interplay of classical and “novel” virulence genes, as well as participation of genes highly conserved in the E. coli species. These studies may inform further rational approaches to vaccine development for these important pathogens. PMID:23892244

  11. Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

    PubMed Central

    Helgesen, Emily; Fossum-Raunehaug, Solveig

    2016-01-01

    ABSTRACT The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a “slow-growth” type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content. IMPORTANCE It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such

  12. Effect of the efflux pump QepA2 combined with chromosomally mediated mechanisms on quinolone resistance and bacterial fitness in Escherichia coli.

    PubMed

    Machuca, Jesús; Briales, Alejandra; Díaz-de-Alba, Paula; Martínez-Martínez, Luis; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2015-09-01

    The aim of the study was to determine the interplay between the plasmid-mediated qepA2 gene and multiple chromosomally mediated fluoroquinolone resistance determinants in the development of fluoroquinolone resistance in Escherichia coli and its influence on bacterial fitness. E. coli ATCC 25922 and derived isogenic strains harbouring different chromosomally mediated fluoroquinolone resistance determinants were electroporated with pBK-CMV vector encoding QepA2. The MICs of fluoroquinolones were determined by standardized microdilution. The mutant prevention concentration (MPC) was evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays. The ciprofloxacin MIC for strains harbouring the qepA2 gene was 4- to 8-fold higher compared with strains without the qepA2 gene. The qepA2 gene also increased the MPC of ciprofloxacin 4- to 16-fold. Combination of the qepA2 gene plus two to three additional mechanisms conferred a clinically relevant resistance level. The presence of the qepA2 gene was associated with fitness costs in strains with mutations in the gyrA and/or parC genes, although the presence of an additional deletion of the marR gene compensated for this fitness cost by increasing bacterial fitness by 5%-23%. The additive effect of chromosomally mediated fluoroquinolone resistance mechanisms and the qepA2 gene led to clinical levels of fluoroquinolone resistance. Under competitive conditions, the qepA2 gene had a biological cost in E. coli that was compensated for by the presence of an additional deletion in the marR gene. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Evolution of carrying capacity in evolution experiments focusing on a single locus on the Escherichia coli chromosome.

    PubMed

    Mori, Kotaro; Kashiwagi, Akiko; Urabe, Itaru; Yomo, Tetsuya

    2009-02-01

    We performed a series of evolution experiments, the results of which illustrated the relationship between mutations and increased carrying capacity (K). Performing an evolution experiment with repeated cycles of mutation by PCR and selection makes it possible to obtain results over shorter culture durations than in methods reported previously relying on spontaneous mutation and selection. We constructed random mutant populations of Escherichia coli in which members differed only in part of the genomic copy of the glutamine synthetase gene and performed daily serial transfer culture where the populations were in K-selected environments. The value of K in this system was increased by 10(5)- to 10(8)-fold relative to the parent clone, which was achieved by four randomly introduced mutations. This method can be applied to any gene and will be useful for analyzing a number of important issues in evolutionary biology.

  14. Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

    PubMed

    Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

    2017-09-11

    The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

  15. Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

    PubMed

    Saxena, Rahul; Vasudevan, Sona; Patil, Digvijay; Ashoura, Norah; Grimwade, Julia E; Crooke, Elliott

    2015-11-24

    DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

  16. Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes.

    PubMed Central

    Lobry, J R; Gautier, C

    1994-01-01

    Multivariate analysis of the amino-acid compositions of 999 chromosome-encoded proteins from Escherichia coli showed that three main factors influence the variability of amino-acid composition. The first factor was correlated with the global hydrophobicity of proteins, and it discriminated integral membrane proteins from the others. The second factor was correlated with gene expressivity, showing a bias in highly expressed genes towards amino-acids having abundant major tRNAs. Just as highly expressed genes have reduced codon diversity in protein coding sequences, so do they have a reduced diversity of amino-acid choice. This showed that translational constraints are important enough to affect the global amino-acid composition of proteins. The third factor was correlated with the aromaticity of proteins, showing that aromatic amino-acid content is highly variable. PMID:8065933

  17. migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

    PubMed Central

    Yamaichi, Yoshiharu; Niki, Hironori

    2004-01-01

    During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization. PMID:14685268

  18. The Chromosomal Arsenic Resistance Genes of Thiobacillus ferrooxidans Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to Escherichia coli

    PubMed Central

    Butcher, Bronwyn G.; Deane, Shelly M.; Rawlings, Douglas E.

    2000-01-01

    The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated. PMID:10788346

  19. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  20. EFFECT OF LYSERGIC ACID DIETHYLAMIDE ON Escherichia coli, STRAIN B/r(lambda).

    DTIC Science & Technology

    LYSERGIC ACIDS , *ESCHERICHIA COLI), GROWTH(PHYSIOLOGY), CHROMOSOMES, DAMAGE, DOSAGE, PURINE ALKALOIDS, ULTRAVIOLET RADIATION, DEOXYRIBONUCLEIC ACIDS , INHIBITION, HALLUCINOGENS, CHEMICAL WARFARE AGENTS, BIOASSAY

  1. Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn Superoxide Dismutases

    PubMed Central

    D'Orazio, Melania; Scotti, Raffaella; Nicolini, Laura; Cervoni, Laura; Rotilio, Giuseppe; Battistoni, Andrea; Gabbianelli, Roberta

    2008-01-01

    Background Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated. Results We report that strains deleted of one or more sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability. Conclusion Our data show that the chromosomal and bacteriophage-associated E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties

  2. Characterization of a chromosomally integrated luxCDABE marker for investigation of shiga toxin-producing Escherichia coli O91:H21 shedding in cattle

    NASA Astrophysics Data System (ADS)

    Hong, Yingying; Mathew, Alan G.

    2011-06-01

    Shiga toxin-producing Escherichia coli (STEC) O91:H21 has been recognized as a potential life-threatening foodborne pathogen and is commonly involved in human infections in European countries. Fecal shedding of the organism by cattle is considered to be the ultimate source for contaminations. Studies examining STEC shedding patterns often include inoculation of strains carrying antibiotic resistance makers for identifiable recovery. However, indigenous intestinal microflora exhibiting similar antibiotic resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves. A chromosomally integrated bioluminescence marker using a luxCDABE cassette from Photorhabdus luminescens was developed in O91:H21 to overcome such shortcomings of antibiotic resistance markers during animal challenge experiment. The marker was validated in various aspects and was shown to have no impact on metabolic reactions, isotype virulence gene patterns, cost to growth, and additionally demonstrated high in vitro stability. Together, the results indicated that a chromosomally integrated luxCDABE based marker may be a superior system for the study of STEC colonization and shedding in cattle.

  3. Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

    SciTech Connect

    Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.; Shanmugam, K.T.; Ingram, L.O. )

    1991-04-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

  4. Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

    PubMed Central

    Rondon, Michelle R.; Raffel, Sandra J.; Goodman, Robert M.; Handelsman, Jo

    1999-01-01

    As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. PMID:10339608

  5. Gravity sensing by Escherichia coli.

    PubMed

    Shimoshige, Hirokazu; Kobayashi, Hideki; Shimamura, Shigeru; Usami, Ron

    2010-01-01

    We investigated the growth and protein profile of Escherichia coli under various gravity strengths to determine the effects of hypergravity on biochemical reactions. E. coli grows at less than 7,500 g without inhibition. Hypergravity induced OmpW and Antigen 43. Changes in gravity strength altered the expression levels of these proteins. This suggests that hypergravity regulates gene expression in bacteria.

  6. Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1.

    PubMed

    Valvano, M A; Silver, R P; Crosa, J H

    1986-04-01

    The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.

  7. Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-streptomyces artificial chromosome vector, pSBAC.

    PubMed

    Liu, Hongbo; Jiang, Hao; Haltli, Bradley; Kulowski, Kerry; Muszynska, Elwira; Feng, Xidong; Summers, Mia; Young, Mairead; Graziani, Edmund; Koehn, Frank; Carter, Guy T; He, Min

    2009-03-27

    Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation, and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.

  8. Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli.

    PubMed

    Tuntufye, Huruma N; Goddeeris, Bruno M

    2011-12-01

    Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Insights Into Mutagenesis Using Escherichia coli Chromosomal lacZ Strains That Enable Detection of a Wide Spectrum of Mutational Events

    PubMed Central

    Seier, Tracey; Padgett, Dana R.; Zilberberg, Gal; Sutera, Vincent A.; Toha, Noor; Lovett, Susan T.

    2011-01-01

    Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3′ exonucleases targeted to the lagging strand. The loss of 3′ exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F′ element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes. PMID:21441210

  10. RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

    PubMed Central

    Tolmachov, Oleg; Palaszewski, Iwona; Bigger, Brian; Coutelle, Charles

    2006-01-01

    Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG

  11. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations

    USDA-ARS?s Scientific Manuscript database

    A novel cloning vector to aid in the construction of ß-galactosidase reporter systems for gene expression studies in lactose metabolizing strains of Shiga toxin producing Escherichia coli is described. The plasmid allows construction of translational fusions of cloned gene promoters with a short seg...

  12. An integrated database to support research on Escherichia coli

    SciTech Connect

    Baehr, A.; Dunham, G.; Matsuda, Hideo; Michaels, G.; Taylor, R.; Overbeek, R.; Rudd, K.E.; Ginsburg, A.; Joerg, D.; Kazic, T.; Hagstrom, R.; Zawada, D.; Smith, C.; Yoshida, Kaoru

    1992-01-01

    We have used logic programming to design and implement a prototype database of genomic information for the model bacterial organism Escherichia coli. This report presents the fundamental database primitives that can be used to access and manipulate data relating to the E. coli genome. The present system, combined with a tutorial manual, provides immediate access to the integrated knowledge base for E. coli chromosome data. It also serves as the foundation for development of more user-friendly interfaces that have the same retrieval power and high-level tools to analyze complex chromosome organization.

  13. Engineering ethanologenic Escherichia coli for levoglucosan utilization.

    PubMed

    Layton, Donovan S; Ajjarapu, Avanthi; Choi, Dong Won; Jarboe, Laura R

    2011-09-01

    Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been shown that recombinant expression of the levoglucosan kinase enzyme enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that existing biocatalysts can be easily modified for levoglucosan utilization. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Iron-requiring mutant of Escherichia coli carrying a deletion in the aroG-nadA region of the chromosome.

    PubMed Central

    Nagy, J; Dénes, G

    1976-01-01

    A mutant of Escherichia coli K-12 carrying a deletion in the aroG-nadA region of the genome requires a high concentration of iron for growth. The strain is chromium sensitive, and in the presence of citrate lower concentrations of iron support cell growth. The deletion mutant lost a gene between aroG and nadA that is responsible for the uptake of iron. PMID:789353

  15. Iron-requiring mutant of Escherichia coli carrying a deletion in the aroG-nadA region of the chromosome.

    PubMed

    Nagy, J; Dénes, G

    1976-10-01

    A mutant of Escherichia coli K-12 carrying a deletion in the aroG-nadA region of the genome requires a high concentration of iron for growth. The strain is chromium sensitive, and in the presence of citrate lower concentrations of iron support cell growth. The deletion mutant lost a gene between aroG and nadA that is responsible for the uptake of iron.

  16. Comparison of clinical categories for Escherichia coli harboring specific qnr and chromosomal-mediated fluoroquinolone resistance determinants according to CLSI and EUCAST.

    PubMed

    Machuca, Jesús; Briales, Alejandra; Díaz-de-Alba, Paula; Martínez-Martínez, Luis; Rodríguez-Martínez, José-Manuel; Pascual, Álvaro

    2016-03-01

    EUCAST breakpoints are more restrictive than those defined by CLSI. This study highlights the discrepancies between CLSI and EUCAST in a well characterized isogenic Escherichia coli collection and their correlations with specific quinolone resistance mechanisms. The greatest number of discrepancies was observed in strains containing 2-4 resistance mechanisms (MIC values on the borderline of clinical resistance). Bearing in mind that quinolones are concentration dependent antimicrobial agents, small changes in MIC may have relevant consequences for treatment outcomes.

  17. Exonuclease IX of Escherichia coli.

    PubMed Central

    Shafritz, K M; Sandigursky, M; Franklin, W A

    1998-01-01

    The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways. PMID:9592142

  18. A gene cluster at an unusual chromosomal location responsible for the novel O-antigen synthesis in Escherichia coli O62 by the ABC transporter-dependent pathway.

    PubMed

    Hou, Xi; Perepelov, Andrei V; Guo, Xi; Senchenkova, Sof'ya N; Shashkov, Alexander S; Liu, Bin; Knirel, Yuriy A; Wang, Lei

    2017-04-10

    The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes.

  19. gltBDF operon of Escherichia coli.

    PubMed Central

    Castaño, I; Bastarrachea, F; Covarrubias, A A

    1988-01-01

    A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega. Images PMID:2448295

  20. Synergistic effects of chromosomal ispB deletion and dxs overexpression on coenzyme Q(10) production in recombinant Escherichia coli expressing Agrobacterium tumefaciens dps gene.

    PubMed

    Choi, Jin-Ho; Ryu, Yeon-Woo; Park, Yong-Cheol; Seo, Jin-Ho

    2009-10-12

    For biotechnological production of coenzyme Q(10) (CoQ(10)) in recombinant Escherichia coli, three genetic manipulations were performed: heterologous expression of decaprenyl diphosphate synthase (Dps) from Agrobacterium tumefaciens, deletion of endogenous octaprenyl diphosphate synthase (IspB), and overexpression of 1-deoxy-d-xylulose synthase (Dxs). Expression of the dps gene and deletion of the ispB gene in E. coli BL21(DE3)DeltaispB/pAP1 allowed production of CoQ(10) only. Furthermore, coexpression of the dxs gene increased the specific content of CoQ(10) from 0.55-0.89mgg(-1) to 1.40mgg(-1). For mass production of CoQ(10), fed-batch fermentation of E. coli BL21(DE3)DeltaispB/pAP1+pDXS was carried out in a defined medium with 20gl(-1) initial glucose and by the glucose-feeding strategy of pH-stat. Finally, 99.4mgl(-1) CoQ(10) concentration, 1.41mgg(-1) specific CoQ(10) content and 3.11mgl(-1)h(-1) productivity were obtained in 33h of the fermentation, which were 78, 1.9, and 19 times higher than those for E. coli BL21(DE3)/pAP1 without the ispB deletion and dxs overexpression.

  1. Robust growth of Escherichia coli.

    PubMed

    Wang, Ping; Robert, Lydia; Pelletier, James; Dang, Wei Lien; Taddei, Francois; Wright, Andrew; Jun, Suckjoon

    2010-06-22

    The quantitative study of the cell growth has led to many fundamental insights in our understanding of a wide range of subjects, from the cell cycle to senescence. Of particular importance is the growth rate, whose constancy represents a physiological steady state of an organism. Recent studies, however, suggest that the rate of elongation during exponential growth of bacterial cells decreases cumulatively with replicative age for both asymmetrically and symmetrically dividing organisms, implying that a "steady-state" population consists of individual cells that are never in a steady state of growth. To resolve this seeming paradoxical observation, we studied the long-term growth and division patterns of Escherichia coli cells by employing a microfluidic device designed to follow steady-state growth and division of a large number of cells at a defined reproductive age. Our analysis of approximately 10(5) individual cells reveals a remarkable stability of growth whereby the mother cell inherits the same pole for hundreds of generations. We further show that death of E. coli is not purely stochastic but is the result of accumulating damages. We conclude that E. coli, unlike all other aging model systems studied to date, has a robust mechanism of growth that is decoupled from cell death.

  2. Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

    PubMed Central

    2013-01-01

    Background Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. Results A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. Conclusions The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal

  3. Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems.

    PubMed

    Grogan, Dennis W; Stengel, Kristy R

    2008-09-01

    In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20-22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2-5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius. The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius, as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.

  4. Peptidoglycan Hydrolases of Escherichia coli

    PubMed Central

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  5. [Virulence mechanisms of enteropathogenic Escherichia coli].

    PubMed

    Farfán-García, Ana Elvira; Ariza-Rojas, Sandra Catherine; Vargas-Cárdenas, Fabiola Andrea; Vargas-Remolina, Lizeth Viviana

    2016-08-01

    Acute diarrheal disease (ADD) is a global public health problem, especially in developing countries and is one of the causes of mortality in children under five. ADD etiologic agents include viruses, bacteria and parasites in that order. Escherichia coli bacteria it is classified as a major diarrheagenic agent and transmitted by consuming contaminated water or undercooked foods. This review compiled updates on information virulence factors and pathogenic mechanisms involved in adhesion and colonization of seven pathotypes of E. coli called enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), shigatoxigenic E. coli (STEC), enteroaggregative E. coli (EAEC) and diffusely-adherent E. coli (DAEC). A final pathotype, adherent-invasive E. coli (AIEC) associated with Crohn's disease was also reviewed. The diarrheagenic pathotypes of E. coli affect different population groups and knowledge of the molecular mechanisms involved in the interaction with the human is important to guide research towards the development of vaccines and new tools for diagnosis and control.

  6. The eclipse period of Escherichia coli

    PubMed Central

    von Freiesleben, Ulrik; Krekling, Martin A.; Hansen, Flemming G.; Løbner-Olesen, Anders

    2000-01-01

    The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be ∼25–30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein’s role in maintaining the eclipse is tied to a function in chromosome organization. PMID:11080169

  7. Independence of replisomes in Escherichia coli chromosomalreplication

    SciTech Connect

    Breier, Adam M.; Weier, Heinz-Ulrich G.; Cozzarelli, Nicholas R.

    2005-03-13

    In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.

  8. Structure of Escherichia coli tryptophanase.

    PubMed

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  9. Structure of Escherichia Coli Tryptophanase

    SciTech Connect

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  10. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli

    PubMed Central

    Jobling, Michael G.

    2016-01-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB5 toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins. PMID:26755534

  11. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli.

    PubMed

    Jobling, Michael G

    2016-04-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB(5) toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins.

  12. Succinate production in Escherichia coli

    PubMed Central

    Thakker, Chandresh; Martínez, Irene; San, Ka-Yiu; Bennett, George N.

    2012-01-01

    Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for production of the four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon that is channeled to the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose as well as other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, as well as the requirement for efficient recovery of succinate and the reliability of the performance under scale-up are important in the overall process. The costs of the overall biorefinery compatible process will determine the economical commercialization of succinate and its impact in larger chemical markets. PMID:21932253

  13. Escherichia coli survival in waters: Temperature dependence

    EPA Science Inventory

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  14. Strategies for Protein Overproduction in Escherichia coli.

    ERIC Educational Resources Information Center

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  15. Strategies for Protein Overproduction in Escherichia coli.

    ERIC Educational Resources Information Center

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  16. Escherichia coli survival in waters: Temperature dependence

    EPA Science Inventory

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  17. Ribitol and D-arabitol catabolism in Escherichia coli.

    PubMed Central

    Scangos, G A; Reiner, A M

    1978-01-01

    In Escherichia coli C, the catabolism of the pentitols ribitol and D-arabitol proceeds through separate, inducible operons, each consisting of a dehydrogenase and a kinase. The ribitol operon is induced in response to ribulose, and the D-arabitol operon is induced in response to D-arabitol. Each operon is under negative control. The genes of the ribitol and D-arabitol operons are very closely linked and lie in a mirror image arrangement, rtlB-rtlA-rtlC-atlC-atlA-atlB, between metG and his on the E. coli chromosome. PMID:350825

  18. Toxigenic Escherichia Coli and Childhood Diarrhea

    PubMed Central

    Mundell, Dave H.; Anselmo, Carl R.; Thrupp, Lauri D.; Wishnow, Rodney M.

    1976-01-01

    Stool specimens were examined from 40 children with diarrhea who were under three years of age to determine the incidence of enterotoxigenic Escherichia coli in endemic diarrhea. Heat-labile E. coli enterotoxin was assayed in the very sensitive and reproducible cultured adrenal tumor cell system. Toxigenic E. coli were isolated from only one stool specimen and in this case infection with Shigella dysenteriae was also present. None of the eight classic enteropathogenic E. coli isolates were positive in the adrenal assay. This study suggests that heat-labile enterotoxin-producing E. coli are not an important cause of endemic childhood diarrhea in Southern California. PMID:775792

  19. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

  20. The roles of Klenow processing and flap processing activities of DNA polymerase I in chromosome instability in Escherichia coli K12 strains.

    PubMed

    Nagata, Yuki; Mashimo, Kazumi; Kawata, Masakado; Yamamoto, Kazuo

    2002-01-01

    The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the DeltapolA and polA107 mutant strains were compared. Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations. The DeltapolA strain, which is deficient in both Klenow domain and 5' --> 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1-4. The polA107 strain, which is deficient in the 5' --> 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2. Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' --> 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4. The 5' --> 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases. We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA.

  1. RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro.

    PubMed Central

    Ogawa, T; Pickett, G G; Kogoma, T; Kornberg, A

    1984-01-01

    Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-base-pair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system. Images PMID:6322184

  2. RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro.

    PubMed

    Ogawa, T; Pickett, G G; Kogoma, T; Kornberg, A

    1984-02-01

    Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-base-pair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system.

  3. Evolution of the iss gene in Escherichia coli.

    PubMed

    Johnson, Timothy J; Wannemuehler, Yvonne M; Nolan, Lisa K

    2008-04-01

    The increased serum survival gene iss has long been recognized for its role in extraintestinal pathogenic Escherichia coli (ExPEC) virulence. iss has been identified as a distinguishing trait of avian ExPEC but not of human ExPEC. This gene has been localized to large virulence plasmids and shares strong similarities with the bor gene from bacteriophage lambda. Here, we demonstrate that three alleles of iss occur among E. coli isolates that appear to have evolved from a common lambda bor precursor. In addition to the occurrence of iss on the ColV/BM virulence plasmids, at least two iss alleles occur within the E. coli chromosome. One of these alleles (designated type 3) was found to occur in the genomes of all currently sequenced ExPEC strains on a similar prophage element that also harbors the Sit iron and manganese transport system. When the prevalence of the three iss types was examined among 487 E. coli isolates, the iss type 3 gene was found to occur at a high frequency among ExPEC isolates, irrespective of the host source. The plasmid-borne iss allele (designated type 1) was highly prevalent among avian pathogenic E. coli and neonatal meningitis-associated E. coli isolates but not among uropathogenic E. coli isolates. This study demonstrates the evolution of iss in E. coli and provides an additional tool for discriminating among E. coli pathotypes through the differentiation of the three iss allele types and bor.

  4. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  5. Completion of DNA replication in Escherichia coli.

    PubMed

    Wendel, Brian M; Courcelle, Charmain T; Courcelle, Justin

    2014-11-18

    The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

  6. Clinical Implications of Enteroadherent Escherichia coli

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2012-01-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including non-intimate adherence mediated by various adhesins. These so called “enteroadherent E. coli ” categories subsequently produced toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  7. Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA

    PubMed Central

    Shaheen, Bashar W.; Nayak, Rajesh; Foley, Steven L.; Boothe, Dawn M.

    2013-01-01

    Objectives To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance. Methods Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses. Results PMQR determinants aac(6′)-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups. Conclusions Novel mutations in topoisomerase genes and PMQR determinants aac(6′)-Ib-cr, qnrS and qepA genes were detected among extended-spectrum β-lactamase-producing E. coli in the USA. PMID:23302578

  8. Inversions between ribosomal RNA genes of Escherichia coli.

    PubMed Central

    Hill, C W; Harnish, B W

    1981-01-01

    It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes. Images PMID:6273909

  9. In-stream Escherichia coli Modeling

    NASA Astrophysics Data System (ADS)

    Pandey, P.; Soupir, M.

    2013-12-01

    Elevated levels of pathogenic bacteria indicators such as Escherichia coli (E. coli) in streams are a serious concern. Controlling E. coli levels in streams requires improving our existing understanding of fate and transport of E. coli at watershed scale. In-stream E. coli concentrations are potentially linked to non-point pollution sources (i.e., agricultural land). Water of a natural stream can receive E. coli by either through overland flow (via runoff from cropland) or resuspension from the streambed to the water column. Calculating in-stream total E. coli loads requires estimation of particle attached bacteria as well free floating E. coli transport. Currently water quality models commonly used for predicting E. coli levels in stream water have limited capability for predicting E. coli levels in the water column as well as in the streambed sediment. The challenges in calculating in-stream E. coli levels include difficulties in modeling the complex interactions between sediment particles and E. coli. Here we have developed a watershed scale model (integrated with Soil and Water Assessment Tool (SWAT)), which involves calculation of particle attached E. coli, to predict in-stream E. coli concentrations. The proposed model predicts E. coli levels in streambed bed sediment as well as in the water column. An extensive in-stream E. coli monitoring was carried out to verify the model predictions, and results indicate that the model performed well. The study proposed here will improve understanding on in-stream bacterial contamination, and help improving existing water quality models for predicting pathogenic bacteria levels in ambient water bodies.

  10. Native valve Escherichia coli endocarditis following urosepsis.

    PubMed

    Rangarajan, D; Ramakrishnan, S; Patro, K C; Devaraj, S; Krishnamurthy, V; Kothari, Y; Satyaki, N

    2013-05-01

    Gram-negative organisms are a rare cause of infective endocarditis. Escherichia coli, the most common cause of urinary tract infection and gram-negative septicemia involves endocardium rarely. In this case report, we describe infection of native mitral valve by E. coli following septicemia of urinary tract origin in a diabetic male; subsequently, he required prosthetic tissue valve replacement indicated by persistent sepsis and congestive cardiac failure.

  11. Extraintestinal pathogenic Escherichia coli: "the other bad E coli".

    PubMed

    Johnson, James R; Russo, Thomas A

    2002-03-01

    Extraintestinal pathogenic Escherichia coli (ExPEC), the specialized strains of E coli that cause most extraintestinal E coli infections, represent a major but little-appreciated health threat. Although the reasons for their evolution remain mysterious, by virtue of their numerous virulence traits ExPEC clearly possess a unique ability to cause disease outside the host intestinal tract. Broader appreciation of the existence and importance of ExPEC and better understandings of their distinctive virulence mechanisms, reservoirs, and transmission pathways may lead to effective preventive interventions against the morbid and costly infections ExPEC cause.

  12. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

  13. Escherichia Coli--Key to Modern Genetics.

    ERIC Educational Resources Information Center

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  14. Detection of O antigens in Escherichia coli

    USDA-ARS?s Scientific Manuscript database

    Lipopolysaccharide on the surface of Escherichia coli constitute the O antigens, which are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in host-pathogen interactions. O antigens that are responsible for antigenic specificity of the ...

  15. Widespread Antisense Transcription in Escherichia coli

    PubMed Central

    Dornenburg, James E.; DeVita, Anne M.; Palumbo, Michael J.; Wade, Joseph T.

    2010-01-01

    ABSTRACT The vast majority of annotated transcripts in bacteria are mRNAs. Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli. We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene. PMID:20689751

  16. Escherichia Coli--Key to Modern Genetics.

    ERIC Educational Resources Information Center

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  17. Escherichia coli and Sudden Infant Death Syndrome

    PubMed Central

    Bettelheim, Karl A.; Goldwater, Paul N.

    2015-01-01

    This review examines the association of strains of Escherichia coli with sudden infant death syndrome (SIDS) and the possible role these bacteria play in this enigmatic condition. The review addresses evidence for E. coli in SIDS infants, potential sources of E. coli in the environment, colonization by commensal and pathogenic strains, the variety of currently accepted pathotypes, and how these pathotypes could compromise intestinal integrity and induce inflammation. Both intestinal and extraintestinal pathotypes are compared in relation to the apparent liability in which virulence traits can be gained or lost by strains of E. coli. The way in which E. coli infections fit with current views on infant sleeping position and other SIDS risk factors is highlighted. PMID:26191064

  18. Cleaving yeast and Escherichia coli genomes at a single site

    SciTech Connect

    Koob, M.; Szybalski, W. )

    1990-10-12

    The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacO{sub s}) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5{prime}-GCGC) methyltransferase (M{center dot}Hha I) in the presence of lac repressor (LacI). All Hae II sites ({prime}-{sub G}{sup A}GCGC{sub C}{sup T}) with the exception of the one in lacO{sub s}, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M{center dot}Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacO{sub s}. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.

  19. Transcription of foreign DNA in Escherichia coli.

    PubMed

    Warren, René L; Freeman, John D; Levesque, Roger C; Smailus, Duane E; Flibotte, Stephane; Holt, Robert A

    2008-11-01

    Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.

  20. Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

    PubMed Central

    Lytle, Darren A.; Rice, Eugene W.; Johnson, Clifford H.; Fox, Kim R.

    1999-01-01

    The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased. PMID:10388724

  1. Characterization of a second lysine decarboxylase isolated from Escherichia coli.

    PubMed Central

    Kikuchi, Y; Kojima, H; Tanaka, T; Takatsuka, Y; Kamio, Y

    1997-01-01

    We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli. PMID:9226257

  2. Hydrogen production by recombinant Escherichia coli strains

    PubMed Central

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  3. Escherichia coli field contamination of pecan nuts.

    PubMed

    Marcus, K A; Amling, H J

    1973-09-01

    More pecan samples collected from grazed orchards were contaminated with Escherichia coli than were samples from nongrazed orchards. No differences in frequency of contamination between mechanically and manually harvested nuts occurred. Nutmeats from whole uncracked pecans that were soaked for 24 h in a lactose broth solution containing E. coli did not become contaminated. Twentyfour percent of the whole pecans soaked in water for 48 h to simulate standing in a rain puddle developed openings along shell suture lines which did not completely close when the nuts were redried.

  4. Uropathogenic Escherichia coli-Associated Exotoxins.

    PubMed

    Welch, Rodney A

    2016-06-01

    Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease.

  5. The evolution of the Escherichia coli phylogeny.

    PubMed

    Chaudhuri, Roy R; Henderson, Ian R

    2012-03-01

    Escherichia coli is familiar to biologists as a classical model system, ubiquitous in molecular biology laboratories around the world. Outside of the laboratory, E. coli strains exist as an almost universal component of the lower-gut flora of humans and animals. Although usually a commensal, E. coli has an alter ego as a pathogen, and is associated with diarrhoeal disease and extra-intestinal infections. The study of E. coli diversity predates the availability of molecular data, with strains initially distinguished by serotyping and metabolic profiling, and genomic diversity illustrated by DNA hybridisation. The quantitative study of E. coli diversity began with the application of multi-locus enzyme electrophoresis (MLEE), and has progressed with the accumulation of nucleotide sequence data, from single genes through multi-locus sequence typing (MLST) to whole genome sequencing. Phylogenetic methods have shed light on the processes of genomic evolution in this extraordinarily diverse species, and revealed the origins of pathogenic E. coli strains, including members of the phylogenetically indistinguishable "genus"Shigella. In May and June 2011, an outbreak of haemorrhagic uraemic syndrome in Germany was linked to a strain of enterohaemorrhagic E. coli (EHEC) O104:H4. Application of high-throughput sequencing technologies allowed the genome and origins of the outbreak strain to be characterised in real time as the outbreak was in progress.

  6. Automatic tracking of Escherichia coli bacteria.

    PubMed

    Xie, Jun; Khan, Shahid; Shah, Mubarak

    2008-01-01

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy videos. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in sequence of frames. Then a novel matching gain measure is introduced to cope with the challenges such as dramatic changes of cells' appearance and serious overlapping and occlusion. For multiple cell tracking, an optimal matching strategy is proposed to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually-determined trajectories, as well as those obtained from existing tracking methods. The stability of the algorithm with different parameter values is also analyzed and discussed.

  7. ELECTRON MICROSCOPY OF PLASMOLYSIS IN ESCHERICHIA COLI.

    PubMed

    COTA-ROBLES, E H

    1963-03-01

    Cota-Robles, Eugene H. (University of California, Riverside). Electron microscopy of plasmolysis in Escherichia coli. J. Bacteriol. 85:499-503. 1963.-Escherichia coli cells plasmolyzed in 0.35 m sucrose reveal plasmolysis at one tip of a cell or in the center of dividing cells in which protoplast partition has been complete. Central plasmolysis reveals that protoplast separation can be completed before the invagination of the cell wall is complete. These studies support the concept that these cells divide by constriction. The strength of the union between cell wall and cytoplasm is not uniform around the entire cell. It is strongest along the sides of these rod-shaped cells and weakest at one tip of the single cell. Thus, a single cell generally forms one cup-shaped vacuole in which the cytoplasm has collapsed away from one tip of the cell.

  8. ELECTRON MICROSCOPY OF PLASMOLYSIS IN ESCHERICHIA COLI

    PubMed Central

    Cota-Robles, Eugene H.

    1963-01-01

    Cota-Robles, Eugene H. (University of California, Riverside). Electron microscopy of plasmolysis in Escherichia coli. J. Bacteriol. 85:499–503. 1963.—Escherichia coli cells plasmolyzed in 0.35 m sucrose reveal plasmolysis at one tip of a cell or in the center of dividing cells in which protoplast partition has been complete. Central plasmolysis reveals that protoplast separation can be completed before the invagination of the cell wall is complete. These studies support the concept that these cells divide by constriction. The strength of the union between cell wall and cytoplasm is not uniform around the entire cell. It is strongest along the sides of these rod-shaped cells and weakest at one tip of the single cell. Thus, a single cell generally forms one cup-shaped vacuole in which the cytoplasm has collapsed away from one tip of the cell. Images PMID:14042923

  9. A new Escherichia coli cell division gene, ftsK.

    PubMed Central

    Begg, K J; Dewar, S J; Donachie, W D

    1995-01-01

    A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer. PMID:7592387

  10. Phage therapy: the Escherichia coli experience.

    PubMed

    Brüssow, Harald

    2005-07-01

    Phages have been proposed as natural antimicrobial agents to fight bacterial infections in humans, in animals or in crops of agricultural importance. Phages have also been discussed as hygiene measures in food production facilities and hospitals. These proposals have a long history, but are currently going through a kind of renaissance as documented by a spate of recent reviews. This review discusses the potential of phage therapy with a specific example, namely Escherichia coli.

  11. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... program for the six non-O157 STEC, as it already does for E. coli O157:H7. The Agency intended to begin... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45...

  12. Systems Metabolic Engineering of Escherichia coli.

    PubMed

    Choi, Kyeong Rok; Shin, Jae Ho; Cho, Jae Sung; Yang, Dongsoo; Lee, Sang Yup

    2017-03-01

    Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

  13. Systems Metabolic Engineering of Escherichia coli.

    PubMed

    Choi, Kyeong Rok; Shin, Jae Ho; Cho, Jae Sung; Yang, Dongsoo; Lee, Sang Yup

    2016-05-01

    Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

  14. Diversity of CRISPR loci in Escherichia coli.

    PubMed

    Díez-Villaseñor, C; Almendros, C; García-Martínez, J; Mojica, F J M

    2010-05-01

    CRISPR (clustered regularly interspaced short palindromic repeats) and CAS (CRISPR-associated sequence) proteins are constituents of a novel genetic barrier that limits horizontal gene transfer in prokaryotes by means of an uncharacterized mechanism. The fundamental discovery of small RNAs as the guides of the defence apparatus arose as a result of Escherichia coli studies. However, a survey of the system diversity in this species in order to further contribute to the understanding of the CRISPR mode of action has not yet been performed. Here we describe two CRISPR/CAS systems found in E. coli, following the analysis of 100 strains representative of the species' diversity. Our results substantiate different levels of activity between loci of both CRISPR types, as well as different target preferences and CRISPR relevances for particular groups of strains. Interestingly, the data suggest that the degeneration of one CRISPR/CAS system in E. coli ancestors could have been brought about by self-interference.

  15. Thymineless death in Escherichia coli: strain specificity.

    PubMed

    Cummings, D J; Mondale, L

    1967-06-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, B(s-12), K-12 rec-21, and possibly K-12 Lon(-), all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation.

  16. Thymineless Death in Escherichia coli: Strain Specificity

    PubMed Central

    Cummings, Donald J.; Mondale, Lee

    1967-01-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, Bs−12, K-12 rec-21, and possibly K-12 Lon−, all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation. Images PMID:5337772

  17. Interaction between Escherichia coli and lunar fines

    NASA Technical Reports Server (NTRS)

    Johansson, K. R.

    1983-01-01

    A sample of mature lunar fines (10084.151) was solubilized to a high degree (about 17 percent) by the chelating agent salicylic acid (0.01. M). The neutralized (pH adjusted to 7.0) leachate was found to inhibit the growth of Escherichia coli (ATCC 259922) in a minimial mineral salts glucose medium; however, the inhibition was somewhat less than that caused by neutralized salicylic acid alone. The presence of lunar fines in the minimal medium was highly stimulatory to growth of E. coli following an early inhibitory response. The bacterium survived less well in the lunar leachate than in distilled water, no doubt because of the salicylate. It was concluded that the sample of lunar soil tested has nutritional value to E. coli and that certain products of fermentation helped to solubilize the lunar soil.

  18. Escherichia coli O 27 in adult diarrhoea.

    PubMed Central

    Hobbs, B. C.; Rowe, B.; Kendall, M.; Turnbull, P. C.; Ghosh, A. C.

    1976-01-01

    Escherichia coli O 27 H 7 was found in 16 stool samples submitted during a Caribbean cruise (Cruise Z) by 29 patients reporting with diarrhoea. A retrospective search revealed E. coli O 27 H 7 in 11 of 20 and 2 of 14 stool cultures from patients on two previous cruises (Y and X respectively) and in a culture from fresh cream (Cruise Y). The repeated occurrence of E. coli O 27 H 7 in the absence of any other apparent cause suggested that this serotype may have been responsible for the diarrhoea. The results of pathogenicity tests suggested that this strain elaborated heat-stable (ST) enterotoxin. The possibility that food may have been the vector is discussed. PMID:794406

  19. Frequency-Dependent Escherichia coli Chemotaxis Behavior

    NASA Astrophysics Data System (ADS)

    Zhu, Xuejun; Si, Guangwei; Deng, Nianpei; Ouyang, Qi; Wu, Tailin; He, Zhuoran; Jiang, Lili; Luo, Chunxiong; Tu, Yuhai

    2012-03-01

    We study Escherichia coli chemotaxis behavior in environments with spatially and temporally varying attractant sources by developing a unique microfluidic system. Our measurements reveal a frequency-dependent chemotaxis behavior. At low frequency, the E. coli population oscillates in synchrony with the attractant. In contrast, in fast-changing environments, the population response becomes smaller and out of phase with the attractant waveform. These observations are inconsistent with the well-known Keller-Segel chemotaxis equation. A new continuum model is proposed to describe the population level behavior of E. coli chemotaxis based on the underlying pathway dynamics. With the inclusion of a finite adaptation time and an attractant consumption rate, our model successfully explains the microfluidic experiments at different stimulus frequencies.

  20. Production of curcuminoids in engineered Escherichia coli.

    PubMed

    Kim, Eun Ji; Cha, Mi Na; Kim, Bog-Gyu; Ahn, Joong-Hoon

    2017-03-09

    Curcumin, a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa, possesses diverse pharmacological properties including anti-inflammatory, antioxidant, antiproliferative and antiangiogenic activity. Two curcuminoids (dicinnamoylmethane and bisdemethoxycurcumin) were synthesized from glucose in Escherichia coli. PAL (phenylalanine ammonia lyase) or TAL (tyrosine ammonia lyase), along with Os4CL (p-coumaroyl-CoA ligase) and CUS (curcumin synthase), were introduced in to E. coli, and each strain produced dicinnamoylmethane or bisdemethoxycurcumin, respectively. In order to increase the production of curcuminoids in E. coli, the shikimic acid biosynthesis pathway which increases the substrates for curcuminoid biosynthesis, was engineered. Using engineered strains, the production of bisdemethoxycurcumin increased from 0.32 to 4.63 mg/L, and that of dicinnamoylmethane from 1.24 mg/L and 6.95 mg/L.

  1. Prodigiosin - A Multifaceted Escherichia coli Antimicrobial Agent

    PubMed Central

    Zorec, Maša; Stopar, David

    2016-01-01

    Despite a considerable interest in prodigiosin, the mechanism of its antibacterial activity is still poorly understood. In this work, Escherichia coli cells were treated with prodigiosin to determine its antimicrobial effect on bacterial physiology. The effect of prodigiosin was concentration dependent. In prodigiosin treated cells above MIC value no significant DNA damage or cytoplasmic membrane disintegration was observed. The outer membrane, however, becomes leaky. Cells had severely decreased respiration activity. In prodigiosin treated cells protein and RNA synthesis were inhibited, cells were elongated but could not divide. Pre-treatment with prodigiosin improved E. coli survival rate in media containing ampicillin, kanamycin and erythromycin but not phleomycin. The results suggest that prodigiosin acts as a bacteriostatic agent in E. coli cells. If prodigiosin was diluted, cells resumed growth. The results indicate that prodigiosin has distinct mode of antibacterial action in different bacteria. PMID:27612193

  2. Inactivation of Escherichia coli by citral.

    PubMed

    Somolinos, M; García, D; Condón, S; Mackey, B; Pagán, R

    2010-06-01

    The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 microl l(-1) of citral at pH 4.0 for 24 h at 20 degrees C caused the inactivation of more than 5 log(10) cycles of cells starting at an inoculum size of 10(6) or 10(7) CFU ml(-1), whereas increasing the cell concentration to 10(9) CFU ml(-1) caused <1 log(10) cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

  3. Cation Transport in Escherichia coli

    PubMed Central

    Schultz, Stanley G.; Solomon, A. K.

    1961-01-01

    Methods have been developed to study the intracellular Na and K concentrations in E. coli, strain K-12. These intracellular cation concentrations have been shown to be functions of the extracellular cation concentrations and the age of the bacterial culture. During the early logarithmic phase of growth, the intracellular K concentration greatly exceeds that of the external medium, whereas the intracellular Na concentration is lower than that of the growth medium. As the age of the culture increases, the intracellular K concentration falls and the intracellular Na concentration rises, changes which are related to the fall in the pH of the medium and to the accumulation of the products of bacterial metabolism. When stationary phase cells, which are rich in Na and poor in K, are resuspended in fresh growth medium, there is a rapid reaccumulation of K and extrusion of Na. These processes represent oppositely directed net ion movements against concentration gradients, and have been shown to be dependent upon the presence of an intact metabolic energy supply. PMID:13909521

  4. Cation Transport in Escherichia coli

    PubMed Central

    Schultz, Stanley G.; Epstein, Wolfgang; Solomon, A. K.

    1963-01-01

    The resuspension of K-poor, Na-rich stationary phase E. coli in fresh medium at pH 7.0 results in a rapid uptake of K and extrusion of Na by the cells. In all experiments net K uptake exceeded net Na extrusion. An investigation of the uptake of glucose, PO4, and Mg and the secretion of H by these cells indicates that the excess K uptake is not balanced by the simultaneous uptake of anions but must be accompanied by the extrusion of cations from the cell. The kinetics of net K uptake are consistent with the existence of two parallel influx processes. The first is rapid, of brief duration, and accounts for approximately 60 per cent of the total net K uptake. This process is a function of the extracellular K concentration, is inhibited in acid media, and appears to be a 1 for 1 exchange of extracellular K for intracellular H. The second influx process has a half-time of approximately 12 minutes, and is not affected by acid media. This process is a function of the intracellular Na concentration, is dependent upon the presence of K in the medium, and may be ascribed to a 1 for 1 exchange of extracellular K for intracellular Na. PMID:14080819

  5. Complete Genome Sequence of Escherichia coli Strain M8, Isolated from ob/ob Mice

    PubMed Central

    Siddharth, Jay; Membrez, Mathieu; Chakrabarti, Anirikh; Betrisey, Bertrand; Chou, Chieh Jason

    2017-01-01

    ABSTRACT Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb. PMID:28572322

  6. Biodegradation of aromatic compounds by Escherichia coli.

    PubMed

    Díaz, E; Ferrández, A; Prieto, M A; García, J L

    2001-12-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications.

  7. Biodegradation of Aromatic Compounds by Escherichia coli

    PubMed Central

    Díaz, Eduardo; Ferrández, Abel; Prieto, María A.; García, José L.

    2001-01-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications. PMID:11729263

  8. Epidemiological and Clinical Complexity of Amoxicillin-Clavulanate-Resistant Escherichia coli

    PubMed Central

    Oteo, Jesús; Ortega, Adriana; Villar, Macarena; Conejo, M. Carmen; Bou, Germán; Aranzamendi-Zaldumbide, Maitane; Cercenado, Emilia; Gurguí, Mercè; Martínez-Martínez, Luis; Merino, María; Rivera, Alba; Oliver, Antonio; Weber, Irene; Pascual, Alvaro; Bartolomé, Rosa M.; Gónzalez-López, Juan José; Campos, José

    2013-01-01

    Two hundred twelve patients with colonization/infection due to amoxicillin-clavulanate (AMC)-resistant Escherichia coli were studied. OXA-1- and inhibitor-resistant TEM (IRT)-producing strains were associated with urinary tract infections, while OXA-1 producers and chromosomal AmpC hyperproducers were associated with bacteremic infections. AMC resistance in E. coli is a complex phenomenon with heterogeneous clinical implications. PMID:23637303

  9. Epidemiological and clinical complexity of amoxicillin-clavulanate-resistant Escherichia coli.

    PubMed

    Rodríguez-Baño, Jesús; Oteo, Jesús; Ortega, Adriana; Villar, Macarena; Conejo, M Carmen; Bou, Germán; Aranzamendi-Zaldumbide, Maitane; Cercenado, Emilia; Gurguí, Mercè; Martínez-Martínez, Luis; Merino, María; Rivera, Alba; Oliver, Antonio; Weber, Irene; Pascual, Alvaro; Bartolomé, Rosa M; Gónzalez-López, Juan José; Campos, José

    2013-07-01

    Two hundred twelve patients with colonization/infection due to amoxicillin-clavulanate (AMC)-resistant Escherichia coli were studied. OXA-1- and inhibitor-resistant TEM (IRT)-producing strains were associated with urinary tract infections, while OXA-1 producers and chromosomal AmpC hyperproducers were associated with bacteremic infections. AMC resistance in E. coli is a complex phenomenon with heterogeneous clinical implications.

  10. Gene controlling L-glutamic acid decarboxylase synthesis in Escherichia coli K-12.

    PubMed

    Lupo, M; Halpern, Y S

    1970-08-01

    Genetically related Escherichia coli K-12 strains were found to differ widely in their l-glutamic acid decarboxylase (GAD) activity. This variation is due to differences in the amount of GAD produced by the different cultures, rather than to the appearance of altered enzymes differing in catalytic activity. A regulatory gene, gadR, which controls the amount of GAD was mapped on the E. coli K-12 chromosome. A strain with a lesion in the structural gene for GAD is described.

  11. Current Interventions for Controlling Pathogenic Escherichia coli.

    PubMed

    Kim, Nam Hee; Cho, Tae Jin; Rhee, Min Suk

    2017-01-01

    This review examined scientific reports and articles published from 2007 to 2016 regarding the major environmental sources of pathogenic Escherichia coli and the routes by which they enter the human gastrointestinal tract. The literature describes novel techniques used to combat pathogenic E. coli transmitted to humans from livestock and agricultural products, food-contact surfaces in processing environments, and food products themselves. Although prevention before contamination is always the best "intervention," many studies aim to identify novel chemical, physical, and biological techniques that inactivate or eliminate pathogenic E. coli cells from breeding livestock, growing crops, and manufactured food products. Such intervention strategies target each stage of the food chain from the perspective of "Farm to Table food safety" and aim to manage major reservoirs of pathogenic E. coli throughout the entire process. Issues related to, and recent trends in, food production must address not only the safety of the food itself but also the safety of those who consume it. Thus, research aims to discover new "natural" antimicrobial agents and to develop "multiple hurdle technology" or other novel technologies that preserve food quality. In addition, this review examines the practical application of recent technologies from the perspective of product quality and safety. It provides comprehensive insight into intervention measures used to ensure food safety, specifically those aimed at pathogenic E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The 503nm pigment of Escherichia coli

    PubMed Central

    Kamitakahara, Joyce R.; Polglase, W. J.

    1970-01-01

    The yield of cell protein was one-third less for streptomycin-dependent Escherichia coli B than for the wild-type parent strain when both were grown aerobically on a medium with limiting glucose, but anaerobically the yield of protein was similar for both strains. The transient pigment absorbing at 503nm that is known to be present in E. coli and other organisms was not detectable in streptomycin-dependent mutants nor in a non-dependent (energy-deficient) revertant. When wild-type E. coli B was grown on limiting glucose–salts medium containing 2,4 dinitrophenol, the yield of cell protein was decreased and formation of the 503nm pigment was inhibited. Fumarase, aconitase and glucose 6-phosphate dehydrogenase were de-repressed in E. coli B cells grown with excess of glucose in a medium containing 2,4-dinitrophenol. In air-oxidized, wild-type E. coli B cells, the 503nm pigment appeared before reduced cytochromes when gluconate was the substrate but failed to appear when succinate was the substrate. The results provide evidence for a role of the 503nm pigment in aerobic energy metabolism, possibly as an electron acceptor from NADPH. PMID:4395501

  13. Redesigning Escherichia coli metabolism for anaerobic production of isobutanol.

    PubMed

    Trinh, Cong T; Li, Johnny; Blanch, Harvey W; Clark, Douglas S

    2011-07-01

    Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an Escherichia coli strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned E. coli strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing E. coli strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.

  14. Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli.

    PubMed

    Todhunter, D A; Smith, K L; Hogan, J S; Nelson, L

    1991-03-01

    Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection. Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E. coli in Freund's Incomplete Adjuvant. At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E. coli (727). Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli. All challenged quarters became infected, and all cows developed acute clinical mastitis. Geometric mean duration of intramammary infections was 6 d for both immunization groups. All infections were spontaneously eliminated within 10 d. No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E. coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI. Reduction in milk production after challenge was greater for cows immunized with E. coli pCRL65/A012. Immunization of dairy cattle with a curli-producing strain of E. coli did not protect against experimental intramammary challenge during lactation.

  15. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  16. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  17. Virulence factors of uropathogenic Escherichia coli.

    PubMed

    Emody, L; Kerényi, M; Nagy, G

    2003-10-01

    Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.

  18. Acid tolerance of enterohemorrhagic Escherichia coli.

    PubMed Central

    Benjamin, M M; Datta, A R

    1995-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains were tested for their ability to survive in acid pH at 37 degrees C. No loss of viability was observed in an O157:H7 EHEC strain (ATCC 43895) at pH levels of 3.0 and 2.5 for at least 5 h. The level of acid tolerance of most EHEC isolates was very high, similar to that of Shigella flexneri strains. The acid tolerance was dependent on the growth phase and pH of the growth medium. PMID:7747983

  19. Detection of Escherichia coli enterotoxins in stools.

    PubMed Central

    Merson, M H; Yolken, R H; Sack, R B; Froehlich, J L; Greenberg, H B; Huq, I; Black, R W

    1980-01-01

    We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method. PMID:6995331

  20. Production of recombinant avidin in Escherichia coli.

    PubMed

    Airenne, K J; Sarkkinen, P; Punnonen, E L; Kulomaa, M S

    1994-06-24

    A recombinant avidin (re-Avd), containing amino acids (aa) 1-123 of the native chicken egg-white Avd, was produced in Escherichia coli. When cells were grown at 37 degrees C production was over 1 microgram/ml, due to altering the codon preference of the first ten codons. The re-Avd was recovered as a soluble protein from cells grown at 25 or 30 degrees C, whereas at 37 degrees C it was mostly insoluble in inclusion bodies. Our results indicated that, despite the potentially harmful biotin-binding activity of Avd, it is possible to produce biologically active Avd in E. coli which then can easily be purified by affinity chromatography on a biotin column in a single step.

  1. Designed phosphoprotein recognition in Escherichia coli.

    PubMed

    Sawyer, Nicholas; Gassaway, Brandon M; Haimovich, Adrian D; Isaacs, Farren J; Rinehart, Jesse; Regan, Lynne

    2014-11-21

    Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

  2. Engineering the Escherichia coli Fermentative Metabolism

    NASA Astrophysics Data System (ADS)

    Orencio-Trejo, M.; Utrilla, J.; Fernández-Sandoval, M. T.; Huerta-Beristain, G.; Gosset, G.; Martinez, A.

    Fermentative metabolism constitutes a fundamental cellular capacity for industrial biocatalysis. Escherichia coli is an important microorganism in the field of metabolic engineering for its well-known molecular characteristics and its rapid growth. It can adapt to different growth conditions and is able to grow in the presence or absence of oxygen. Through the use of metabolic pathway engineering and bioprocessing techniques, it is possible to explore the fundamental cellular properties and to exploit its capacity to be applied as industrial biocatalysts to produce a wide array of chemicals. The objective of this chapter is to review the metabolic engineering efforts carried out with E. coli by manipulating the central carbon metabolism and fermentative pathways to obtain strains that produce metabolites with high titers, such as ethanol, alanine, lactate and succinate.

  3. Escherichia coli growth under modeled reduced gravity

    NASA Technical Reports Server (NTRS)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  4. Escherichia coli growth under modeled reduced gravity

    NASA Technical Reports Server (NTRS)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  5. Transport proteins promoting Escherichia coli pathogenesis.

    PubMed

    Tang, Fengyi; Saier, Milton H

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

  6. Engineering Escherichia coli for methanol conversion.

    PubMed

    Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J; Wendisch, Volker F; Brautaset, Trygve; Portais, Jean-Charles; Vorholt, Julia A

    2015-03-01

    Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

  7. Transport proteins promoting Escherichia coli pathogenesis

    PubMed Central

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  8. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

  9. Engineering Escherichia coli to bind to cyanobacteria.

    PubMed

    Zhang, Zijian; Meng, Liuyi; Ni, Congjian; Yao, Lanqiu; Zhang, Fengyu; Jin, Yuji; Mu, Xuelang; Zhu, Shiyu; Lu, Xiaoyu; Liu, Shiyu; Yu, Congyu; Wang, Chenggong; Zheng, Pu; Wu, Jie; Kang, Li; Zhang, Haoqian M; Ouyang, Qi

    2017-03-01

    We engineered Escherichia coli cells to bind to cyanobacteria by heterologously producing and displaying lectins of the target cyanobacteria on their surface. To prove the efficacy of our approach, we tested this design on Microcystis aeruginosa with microvirin (Mvn), the lectin endogenously produced by this cyanobacterium. The coding sequence of Mvn was C-terminally fused to the ice nucleation protein NC (INPNC) gene and expressed in E. coli. Results showed that E. coli cells expressing the INPNC::Mvn fusion protein were able to bind to M. aeruginosa and the average number of E. coli cells bound to each cyanobacterial cell was enhanced 8-fold. Finally, a computational model was developed to simulate the binding reaction and help reconstruct the binding parameters. To our best knowledge, this is the first report on the binding of two organisms in liquid culture mediated by the surface display of lectins and it may serve as a novel approach to mediate microbial adhesion.

  10. Inactivation of Escherichia coli by ultrasonic irradiation.

    PubMed

    Furuta, M; Yamaguchi, M; Tsukamoto, T; Yim, B; Stavarache, C E; Hasiba, K; Maeda, Y

    2004-04-01

    Ultrasonic inactivation of Escherichia coli XL1-Blue has been investigated by high-intensity ultrasonic waves from horn type sonicator (27.5 kHz) utilizing the "squeeze-film effect". The amplitude of the vibration face contacting the sample solution was used as an indication of the ultrasonic power intensity. The inactivation of the E. coli cells by ultrasonic irradiation shows pseudo first-order behavior. The inactivation rate constant gradually increased with increasing amplitude of the vibration face and showed rapid increase above 3 microm (p-p). In contrast, the H2O2 formation was not observed below 3 microm (p-p), indicating that the ultrasonic shock wave might be more important than indirect effect of OH radicals formed by ultrasonic cavitation in this system. The optimal thickness of the squeeze film was determined as 2 mm for the E. coli inactivation. More than 99% of E. coli cells was inactivated within 180-s sonication at the amplitude of 3 microm (p-p) and 2 mm of the thickness of the squeeze film.

  11. Compilation of DNA sequences of Escherichia coli

    PubMed Central

    Kröger, Manfred

    1989-01-01

    We have compiled the DNA sequence data for E.coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature. We have introduced all available genetic map data and have arranged the sequences accordingly. As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989. This corresponds to a total of 19.92% of the entire E.coli chromosome consisting of about 4,720 kbp. This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences. This compilation may be available in machine readable form from one of the international databanks in some future. PMID:2654890

  12. Enteroaggregative Escherichia coli an emergent pathogen with different virulence properties.

    PubMed

    Villaseca, J M; Hernández, U; Sainz-Espuñes, T R; Rosario, C; Eslava, C

    2005-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emergent bacterial pathogen. The first studies in developing countries with EAEC strains, showed that this bacterium was associated with persistent diarrhea. However, new studies showed that EAEC may be associated also with acute diarrhea, with both nosocomial and community outbreaks worldwide, and as an important pathogen of diarrheal disease in human immunodeficiency virus-infected adults. EAEC strains are recognized by their characteristic aggregative adherence or "stacked-brick" pattern to epithelial cells. Although the pathogenesis of EAEC infection is not well understood, cellular changes observed in animal models and in vitro assays, suggested that the alterations in the intestinal mucosa during EAEC infection are associated with adherence factors and toxins production. The damage has been associated with the release of inflammatory mediators, which may contribute also to the intestinal illness. The dissemination of the high pathogenicity island from Yersinia pestis evolutionary group to EAEC has been show; different studies suggest that it may contribute to the virulence of EAEC strains. Molecular methods to investigate the presence of plasmid and chromosomal EAEC-associated virulence markers, have been used for the characterization and epidemiological studies of EAEC strains. Although the clinical and epidemiological importance of EAEC have been demonstrated in different studies, Escherichia coli strains with adherent agreggative phenotype are commonly isolated from healthy children and environmental sources. This support the necessity to study virulence factors no related with the cells adherence pattern, that show the specific EAEC pathogenic clones associated whit intestinal disease.

  13. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Escherichia coli serological reagents. 866.3255 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... antisera conjugated with a fluorescent dye used to identify Escherichia coli directly from...

  14. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Escherichia coli serological reagents. 866.3255 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... antisera conjugated with a fluorescent dye used to identify Escherichia coli directly from...

  15. Susceptibilities of Escherichia coli and Staphylococcus aureus to Aloe barbadensis.

    PubMed

    Shilpakala, S R; Prathiba, J; Malathi, R

    2009-01-01

    The in vitro susceptibilities of Escherichia coli and Staphylococcus aureus were evaluated and the two organisms were susceptible to the inner gel of aloe barbadensis, though it was more effective against Staphylococcus aureus than Escherichia coli. The reduction for Aloe Vera (AV) needed to suppress the growth of the gram-positive bacterium was attributed to the structural differences between the two organisms.

  16. A series of template plasmids for Escherichia coli genome engineering.

    PubMed

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

    PubMed Central

    Zeng, H.; Snavely, I.; Zamorano, P.; Javor, G. T.

    1998-01-01

    Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb− phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery. PMID:9658014

  18. Novel Antigens for enterotoxigenic Escherichia coli (ETEC) Vaccines

    PubMed Central

    Fleckenstein, James M.; Sheikh, Alaullah; Qadri, Firdausi

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial pathogens-causing diarrhea in developing countries where they cause hundreds of thousands of deaths, mostly in children. These organisms are leading cause of diarrheal illness in travelers to endemic countries. ETEC pathogenesis, and consequently vaccine approaches, have largely focused on plasmid-encoded enterotoxins or fimbrial colonization factors. To date these approaches have not yielded a broadly protective vaccine. However, recent studies suggest that ETEC pathogenesis is more complex than previously appreciated and involves additional plasmid and chromosomally-encoded virulence molecules that can be targeted in vaccines. Here, we review recent novel antigen discovery efforts, potential contribution of these proteins to the molecular pathogenesis of ETEC and protective immunity, and the potential implications for development of next generation vaccines for important pathogens. These proteins may help to improve the effectiveness of future vaccines by making simpler and possibly broadly protective because of their conserved nature. PMID:24702311

  19. Effects of Toluene on Escherichia coli

    PubMed Central

    Jackson, Robert W.; DeMoss, J. A.

    1965-01-01

    Jackson, Robert W. (University of California, San Diego, La Jolla), and J. A. DeMoss. Effects of toluene on Escherichia coli. J. Bacteriol. 90:1420–1425. 1965.—When toluene is added at appropriate levels to exponentially growing cultures of Escherichia coli, a time-dependent loss of turbidity is observed which is concurrent with a loss of material to the medium and with unmasking of β-galactosidase. In addition, the galactoside permease system is totally destroyed. Electron micrographs confirm the indications that the cells are not being lysed by toluene, although the cytoplasm collapses to the interior of the cell. Included in the material lost from the cell after toluene treatment is 85% of the total ribonucleic acid (RNA), the principal source of which appears to be the ribosomes. The loss of RNA is temperature-dependent. Protein is also lost to the medium as a function of both temperature and available toluene. Up to 25% of the total protein is found in the medium, the precise amount depending on the level of toluene employed. Zone centrifugation studies of extracts from treated cells indicate that toluene elicits a rapid disaggregation of ribosomes that is terminated, at any stage, by disruption of the cells. The disaggregation is temperature-dependent and does not occur at 4 C. It appears to be distinct from the actual degradation of ribosomal RNA and is accompanied by an accumulation of small particles during the initial phases of treatment at 21 C. Toluene added to crude extracts of normal E. coli cells is unable to cause detectable ribosome destruction. Images PMID:5321488

  20. Transfection of Escherichia coli spheroplasts: infectious lambda prophage DNA.

    PubMed

    Benzinger, R; Kleber, I; Huskey, R

    1978-06-01

    High mol. wt. DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious. Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E. coli (lambda c857 tsxisam6) lysogen yielded 200 times as many plaques on sup+ than on sup- spheroplasts. Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not. About 10(4) genome equivalents of E. coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments.

  1. DNA sequence of the Escherichia coli tonB gene.

    PubMed Central

    Postle, K; Good, R F

    1983-01-01

    The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the tonB gene has been determined. Transcription initiation and termination sites for tonB RNA have been determined by S1 nuclease mapping. The tonB promoter and terminator resemble other E. coli promoters and terminators; the sequence of the tonB terminator region suggests that it may function bidirectionally. The DNA sequence specifies an open translation reading frame between the 5' and 3' RNA termini whose location is consistent with the position of previously isolated tonB::IS1 mutations. The DNA sequence predicts a proline-rich protein with a calculated size of 26.1-26.6 kilodaltons (239-244 amino acids), depending on which of three potential initiation codons is utilized. The predicted NH2 terminus of tonB protein resembles the proteolytically cleaved signal sequences of E. coli periplasmic and outer membrane proteins; the overall hydrophilic character of the protein sequence suggests that the bulk of the tonB protein is not embedded within the inner or outer membrane. A significant discrepancy exists between the calculated size of tonB protein and the apparent size of 36 kilodaltons determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Images PMID:6310567

  2. Operons in Escherichia coli: genomic analyses and predictions.

    PubMed

    Salgado, H; Moreno-Hagelsieb, G; Smith, T F; Collado-Vides, J

    2000-06-06

    The rich knowledge of operon organization in Escherichia coli, together with the completed chromosomal sequence of this bacterium, enabled us to perform an analysis of distances between genes and of functional relationships of adjacent genes in the same operon, as opposed to adjacent genes in different transcription units. We measured and demonstrated the expected tendencies of genes within operons to have much shorter intergenic distances than genes at the borders of transcription units. A clear peak at short distances between genes in the same operon contrasts with a flat frequency distribution of genes at the borders of transcription units. Also, genes in the same operon tend to have the same physiological functional class. The results of these analyses were used to implement a method to predict the genomic organization of genes into transcription units. The method has a maximum accuracy of 88% correct identification of pairs of adjacent genes to be in an operon, or at the borders of transcription units, and correctly identifies around 75% of the known transcription units when used to predict the transcription unit organization of the E. coli genome. Based on the frequency distance distributions, we estimated a total of 630 to 700 operons in E. coli. This step opens the possibility of predicting operon organization in other bacteria whose genome sequences have been finished.

  3. Global regulation of gene expression in Escherichia coli.

    PubMed Central

    Chuang, S E; Daniels, D L; Blattner, F R

    1993-01-01

    Global transcription responses of Escherichia coli to various stimuli or genetic defects were studied by measuring mRNA levels in about 400 segments of the genome. Measuring mRNA levels was done by analyzing hybridization to DNA dot blots made with overlapping lambda clones spanning the genome of E. coli K-12. Conditions examined included isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, heat shock, osmotic shock, starvation for various nutrients, entrance of cells into the stationary phase of growth, anaerobic growth in a tube, growth in the gnotobiotic mouse gut, and effects of pleiotropic mutations rpoH, himA, topA, and crp. Most mapped genes known to be regulated by a particular situation were successfully detected. In addition, many chromosomal regions containing no previously known regulated genes were discovered that responded to various stimuli. This new method for studying globally regulated genetic systems in E. coli combines detection, cloning, and physical mapping of a battery of coregulated genes in one step. Images PMID:8458845

  4. The gene-protein database of Escherichia coli: edition 5.

    PubMed

    VanBogelen, R A; Sankar, P; Clark, R L; Bogan, J A; Neidhardt, F C

    1992-12-01

    The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.

  5. Comparison of 61 Sequenced Escherichia coli Genomes

    PubMed Central

    Lukjancenko, Oksana; Wassenaar, Trudy M.

    2010-01-01

    Escherichia coli is an important component of the biosphere and is an ideal model for studies of processes involved in bacterial genome evolution. Sixty-one publically available E. coli and Shigella spp. sequenced genomes are compared, using basic methods to produce phylogenetic and proteomics trees, and to identify the pan- and core genomes of this set of sequenced strains. A hierarchical clustering of variable genes allowed clear separation of the strains into clusters, including known pathotypes; clinically relevant serotypes can also be resolved in this way. In contrast, when in silico MLST was performed, many of the various strains appear jumbled and less well resolved. The predicted pan-genome comprises 15,741 gene families, and only 993 (6%) of the families are represented in every genome, comprising the core genome. The variable or ‘accessory’ genes thus make up more than 90% of the pan-genome and about 80% of a typical genome; some of these variable genes tend to be co-localized on genomic islands. The diversity within the species E. coli, and the overlap in gene content between this and related species, suggests a continuum rather than sharp species borders in this group of Enterobacteriaceae. PMID:20623278

  6. Role of Escherichia coli in Biofuel Production.

    PubMed

    Koppolu, Veerendra; Vasigala, Veneela Kr

    2016-01-01

    Increased energy consumption coupled with depleting petroleum reserves and increased greenhouse gas emissions have renewed our interest in generating fuels from renewable energy sources via microbial fermentation. Central to this problem is the choice of microorganism that catalyzes the production of fuels at high volumetric productivity and yield from cheap and abundantly available renewable energy sources. Microorganisms that are metabolically engineered to redirect renewable carbon sources into desired fuel products are contemplated as best choices to obtain high volumetric productivity and yield. Considering the availability of vast knowledge in genomic and metabolic fronts, Escherichia coli is regarded as a primary choice for the production of biofuels. Here, we reviewed the microbial production of liquid biofuels that have the potential to be used either alone or in combination with the present-day fuels. We specifically highlighted the metabolic engineering and synthetic biology approaches used to improve the production of biofuels from E. coli over the past few years. We also discussed the challenges that still exist for the biofuel production from E. coli and their possible solutions.

  7. Role of Escherichia coli in Biofuel Production

    PubMed Central

    Koppolu, Veerendra; Vasigala, Veneela KR

    2016-01-01

    Increased energy consumption coupled with depleting petroleum reserves and increased greenhouse gas emissions have renewed our interest in generating fuels from renewable energy sources via microbial fermentation. Central to this problem is the choice of microorganism that catalyzes the production of fuels at high volumetric productivity and yield from cheap and abundantly available renewable energy sources. Microorganisms that are metabolically engineered to redirect renewable carbon sources into desired fuel products are contemplated as best choices to obtain high volumetric productivity and yield. Considering the availability of vast knowledge in genomic and metabolic fronts, Escherichia coli is regarded as a primary choice for the production of biofuels. Here, we reviewed the microbial production of liquid biofuels that have the potential to be used either alone or in combination with the present-day fuels. We specifically highlighted the metabolic engineering and synthetic biology approaches used to improve the production of biofuels from E. coli over the past few years. We also discussed the challenges that still exist for the biofuel production from E. coli and their possible solutions. PMID:27441002

  8. Microbubble assisted polyhydroxybutyrate production in Escherichia coli.

    PubMed

    Inan, Kadriye; Sal, Fulya Ay; Rahman, Asif; Putman, Ryan J; Agblevor, Foster A; Miller, Charles D

    2016-07-09

    One of the potential limitations of large scale aerobic Escherichia coli fermentation is the need for increased dissolved oxygen for culture growth and bioproduct generation. As culture density increases the poor solubility of oxygen in water becomes one of the limiting factors for cell growth and product formation. A potential solution is to use a microbubble dispersion (MBD) generating device to reduce the diameter and increase the surface area of sparged bubbles in the fermentor. In this study, a recombinant E. coli strain was used to produce polyhydroxybutyrate (PHB) under conventional and MBD aerobic fermentation conditions. In conventional fermentation operating at 350 rpm and 0.8 vvm air flow rate, an OD600 of 6.21 and PHB yield of 23 % (dry cell basis) was achieved. MBD fermentation with similar bioreactor operating parameters produced an OD600 of 8.17 and PHB yield of 43 % PHB, which was nearly double that of the conventional fermentation. This study demonstrated that using a MBD generator can increase oxygen mass transfer into the aqueous phase, increasing E. coli growth and bioproduct generation.

  9. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1989-01-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for fermentation and anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its cloning sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting the ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  10. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The purpose of this project is to elucidate the way in which the synthesis of ethanol and related fermentation products are regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its coding sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase and have recently cloned the ldh gene. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  11. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  12. Long term effects of Escherichia coli mastitis.

    PubMed

    Blum, Shlomo E; Heller, Elimelech D; Leitner, Gabriel

    2014-07-01

    Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by <15% decrease in DMY and <30 days until return to normal (n = 5), and 'long inflammation', characterized by >15% decrease in DMY and >30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

  13. WGS accurately predicts antimicrobial resistance in Escherichia coli

    USDA-ARS?s Scientific Manuscript database

    Objectives: To determine the effectiveness of whole-genome sequencing (WGS) in identifying resistance genotypes of multidrug-resistant Escherichia coli (E. coli) and whether these correlate with observed phenotypes. Methods: Seventy-six E. coli strains were isolated from farm cattle and measured f...

  14. Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

    PubMed Central

    Siegel, Eli C.

    1973-01-01

    An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage λ. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut+ strains. UV irradiation induced mutations in a mutU4 strain, and phage λ was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4. PMID:4345920

  15. Short-time movement of E. coli chromosomal loci depends on coordinate and subcellular localization.

    PubMed

    Javer, Avelino; Long, Zhicheng; Nugent, Eileen; Grisi, Marco; Siriwatwetchakul, Kamin; Dorfman, Kevin D; Cicuta, Pietro; Cosentino Lagomarsino, Marco

    2013-01-01

    In bacteria, chromosomal architecture shows strong spatial and temporal organization, and regulates key cellular functions, such as transcription. Tracking the motion of chromosomal loci at short timescales provides information related to both the physical state of the nucleo-protein complex and its local environment, independent of large-scale motions related to genome segregation. Here we investigate the short-time (0.1-10 s) dynamics of fluorescently labelled chromosomal loci in Escherichia coli at different growth rates. At these timescales, we observe for the first time a dependence of the loci's apparent diffusion on both their subcellular localization and chromosomal coordinate, and we provide evidence that the properties of the chromosome are similar in the tested growth conditions. Our results indicate that either non-equilibrium fluctuations due to enzyme activity or the organization of the genome as a polymer-protein complex vary as a function of the distance from the origin of replication.

  16. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed Central

    Adler, H; Mural, R; Suttle, B

    1992-01-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. Images PMID:1551829

  17. An overview of atypical enteropathogenic Escherichia coli.

    PubMed

    Hernandes, Rodrigo T; Elias, Waldir P; Vieira, Mônica A M; Gomes, Tânia A T

    2009-08-01

    The enteropathogenic Escherichia coli (EPEC) pathotype is currently divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that distinguishes these two groups is the presence of the EPEC adherence factor plasmid, which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. Herein, we review the serotypes, virulence properties, genetic relationships, epidemiology, reservoir and diagnosis of aEPEC, including those strains not belonging to the classical EPEC serogroups (nonclassical EPEC serogroups). The large variety of serotypes and genetic virulence properties of aEPEC strains from nonclassical EPEC serogroups makes it difficult to determine which strains are truly pathogenic.

  18. Mechanism of Escherichia coli Resistance to Pyrrhocoricin

    PubMed Central

    Narayanan, Shalini; Modak, Joyanta K.; Ryan, Catherine S.; Garcia-Bustos, Jose; Davies, John K.

    2014-01-01

    Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10−7. Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials. PMID:24590485

  19. Escherichia coli fliAZY operon.

    PubMed Central

    Mytelka, D S; Chamberlin, M J

    1996-01-01

    We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3. PMID:8550423

  20. Animal models of enteroaggregative Escherichia coli infection

    PubMed Central

    Philipson, Casandra W.; Bassaganya-Riera, Josep; Hontecillas, Raquel

    2013-01-01

    Enteroaggregative Escherichia coli (EAEC) has been acknowledged as an emerging cause of gastroenteritis worldwide for over two decades. Epidemiologists are revealing the role of EAEC in diarrheal outbreaks as a more common occurrence than ever suggested before. EAEC induced diarrhea is most commonly associated with travelers, children and immunocompromised individuals however its afflictions are not limited to any particular demographic. Many attributes have been discovered and characterized surrounding the capability of EAEC to provoke a potent pro-inflammatory immune response, however cellular and molecular mechanisms underlying initiation, progression and outcomes are largely unknown. This limited understanding can be attributed to heterogeneity in strains and the lack of adequate animal models. This review aims to summarize current knowledge about EAEC etiology, pathogenesis and clinical manifestation. Additionally, current animal models and their limitations will be discussed along with the value of applying systems-wide approaches such as computational modeling to study host-EAEC interactions. PMID:23680797

  1. Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

    PubMed Central

    Juhas, Mario; Wong, Christine; Ajioka, James W.

    2016-01-01

    The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis. PMID:27798678

  2. Genetic transformation in Escherichia coli K12.

    PubMed

    Cosloy, S D; Oishi, M

    1973-01-01

    An auxotrophic strain of E. coli K12 treated with CaCl(2) was transformed for several markers at a frequency of up to 10(-6) per recipient cell by a DNA preparation isolated from a prototrophic strain. The transforming activity of the DNA preparation was eliminated by treatment with DNase, heat, or sonication, whereas RNase or Pronase treatment had little effect. Two closely linked genetic markers (leu and ara) showed a high degree of cotransformation linkage when high molecular weight DNA was used, but the linkage was almost completely eliminated when sheared, smaller molecular weight DNA was used. There is genetic evidence that the transformation is a result of the replacement of the preexisting genetic marker on the chromosome by that of the donor DNA.

  3. The thermal impulse response of Escherichia coli

    PubMed Central

    Paster, Eli; Ryu, William S.

    2008-01-01

    Swimming Escherichia coli responds to changes in temperature by modifying its motor behavior. Previous studies using populations of cells have shown that E. coli accumulate in spatial thermal gradients, but these experiments did not cleanly separate thermal responses from chemotactic responses. Here we have isolated the thermal response by studying the behavior of single, tethered cells. The motor output of cells grown at 33°C was measured at constant temperature, from 10° to 40°C, and in response to small, impulsive increases in temperature, from 23° to 43°C. The thermal impulse response at temperatures < 31°C is similar to the chemotactic impulse response: Both follow a similar time course, share the same directionality, and show biphasic characteristics. At temperatures > 31°C, some cells show an inverted response, switching from warm- to cold-seeking behavior. The fraction of inverted responses increases nonlinearly with temperature, switching steeply at the preferred temperature of 37°C. PMID:18385380

  4. Characterization of enterotoxigenic bovine Escherichia coli.

    PubMed Central

    Sivaswamy, G; Gyles, C L

    1976-01-01

    Among 300 isolates of bovine Escherichia coli, 56 which had been found enterotoxigenic in calf gut loops were characterized on the basis of O and K antigens, colonial morphology and resistance to seven antimicrobial drugs. The 56 isolates enterotoxigenic in the calf were compared with the nonenterotoxigenic ones. Of the 56 enterotoxigenic E. coli the majority possessed the A type of K antigen and had OK groups, O9:K(PS274) or O101:K(RVC118). Fourteen of these isolates had the K99 antigen. None of 27 isolates found enterotoxigenic in the piglet but not in the calf possessed the K99 antigen or belonged to OK groups O9:K(PS274) or O101:K(RVC118). Comparison of the patterns of resistance to seven antimicrobial drugs showed that all enterotoxigenic and nonenterotoxigenic isolates were susceptible to nitrofurantoin and sulphachlorphyridiazine and that there was no significant difference in the patterns between the two groups. The majority of enterotoxigenic isolates were mucoid, whereas most of the nonenterotoxigenic isolates were nonmucoid. PMID:793694

  5. The crystal structure Escherichia coli Spy

    PubMed Central

    Kwon, Eunju; Kim, Dong Young; Gross, Carol A; Gross, John D; Kim, Kyeong Kyu

    2010-01-01

    Escherichia coli spheroplast protein y (EcSpy) is a small periplasmic protein that is homologous with CpxP, an inhibitor of the extracytoplasmic stress reponse. Stress conditions such as spheroplast formation induce the expression of Spy via the Cpx or the Bae two-component systems in E. coli, though the function of Spy is unknown. Here, we report the crystal structure of EcSpy, which reveals a long kinked hairpin-like structure of four α-helices that form an antiparallel dimer. The dimer contains a curved oval shape with a highly positively charged concave surface that may function as a ligand binding site. Sequence analysis reveals that Spy is highly conserved over the Enterobacteriaceae family. Notably, three conserved regions that contain identical residues and two LTxxQ motifs are placed at the horizontal end of the dimer structure, stablizing the overall fold. CpxP also contains the conserved sequence motifs and has a predicted secondary structure similar to Spy, suggesting that Spy and CpxP likely share the same fold. PMID:20799348

  6. The crystal structure Escherichia coli Spy.

    PubMed

    Kwon, Eunju; Kim, Dong Young; Gross, Carol A; Gross, John D; Kim, Kyeong Kyu

    2010-11-01

    Escherichia coli spheroplast protein y (EcSpy) is a small periplasmic protein that is homologous with CpxP, an inhibitor of the extracytoplasmic stress response. Stress conditions such as spheroplast formation induce the expression of Spy via the Cpx or the Bae two-component systems in E. coli, though the function of Spy is unknown. Here, we report the crystal structure of EcSpy, which reveals a long kinked hairpin-like structure of four α-helices that form an antiparallel dimer. The dimer contains a curved oval shape with a highly positively charged concave surface that may function as a ligand binding site. Sequence analysis reveals that Spy is highly conserved over the Enterobacteriaceae family. Notably, three conserved regions that contain identical residues and two LTxxQ motifs are placed at the horizontal end of the dimer structure, stabilizing the overall fold. CpxP also contains the conserved sequence motifs and has a predicted secondary structure similar to Spy, suggesting that Spy and CpxP likely share the same fold.

  7. Chemotaxis Toward Sugars in Escherichia coli

    PubMed Central

    Adler, Julius; Hazelbauer, Gerald L.; Dahl, M. M.

    1973-01-01

    Using a quantitative assay for measuring chemotaxis, we tested a variety of sugars and sugar derivatives for their ability to attract Escherichia coli bacteria. The most effective attractants, i.e., those that have thresholds near 10−5 M or below, are N-acetyl-d-glucosamine, 6-deoxy-d-glucose, d-fructose, d-fucose, 1-d-glycerol-β-d-galactoside, galactitol, d-galactose, d-glucosamine, d-glucose, α-d-glucose-1-phosphate, lactose, maltose, d-mannitol, d-mannose, methyl-β-d-galactoside, methyl-β-d-glucoside, d-ribose, d-sorbitol, and trehalose. Lactose, and probably d-glucose-1-phosphate, are attractive only after conversion to the free monosaccharide, while the other attractants do not require breakdown for taxis. Nine different chemoreceptors are involved in detecting these various attractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose, maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificity of each was studied. The chemoreceptors, with the exception of the one for d-glucose, are inducible. The galactose-binding protein serves as the recognition component of the galactose chemoreceptor. E. coli also has osmotically shockable binding activities for maltose and d-ribose, and these appear to serve as the recognition components for the corresponding chemoreceptors. PMID:4580570

  8. Expanding ester biosynthesis in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2015-01-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l−1). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters. PMID:24609358

  9. [Enteroinvasive Escherichia coli. Pathogenesis and epidemiology].

    PubMed

    Prats, G; Llovet, T

    1995-03-01

    Enteroinvasive Escherichia coli (EIEC) is an intestinal pathogen causing enteritis, with a similar pathogenic mechanism to that of Shigella, which causes an epithelial invasion of the large bowel leading to inflammation and ulceration of the mucosa. The patients often develop the symptoms of bacillary dysentery. The EIEC strains are atypical in their biochemical reactions and may ferment lactose late or not at all, are lysine decarboxilase negative, and non motile. In addition, most EIEC strains express somatic antigens which are either strongly related or identical to Shigella antigens. EIEC invasion is mediated by a large plasmid (140 MDa) coding for the production of several outer membrane proteins involved in invasiveness. These strains have been isolated with some regularity in South America, the Extreme Orient, and Eastern Europe. In Spain the incidence of enteroinvasive E. coli is extraordinarily low (0.2%), the serogroup O124 being the most frequently isolated. EIEC enteritis has been associated to sporadic cases occurring in travellers. Occasional outbreaks related to ingestion of contaminated water or food and person to person have been reported.

  10. Isobutanol production from cellobiose in Escherichia coli.

    PubMed

    Desai, Shuchi H; Rabinovitch-Deere, Christine A; Tashiro, Yohei; Atsumi, Shota

    2014-04-01

    Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64 ± 0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.

  11. RESISTANCE OF ESCHERICHIA COLI TO TETRACYCLINES.

    PubMed

    FRANKLIN, T J; GODFREY, A

    1965-01-01

    1. A strain of Escherichia coli highly resistant to chlortetracycline and partially cross-resistant to tetracycline has been isolated. 2. The nitro-reductase system of the resistant cells was inhibited to a smaller extent by chlortetracycline than was the corresponding enzyme of sensitive cells. 3. The incorporation of leucine in vitro into the ribosomal protein of cell-free preparations from sensitive and resistant cells was equally inhibited by chlortetracycline. 4. Resistant cells accumulated much less chlortetracycline and tetracycline than did sensitive cells when both were cultured in the presence of these drugs. 5. The uptake of tetracycline by both sensitive and resistant E. coli was dependent on the presence of glucose in the medium. 6. Fractionation of cells cultured in medium containing [(14)C]chlortetracycline indicated that the largest proportion of radioactivity in sensitive cells was in the fraction consisting mainly of cell-wall material. There was no concentration of radioactivity in any one fraction of the resistant cells. 7. No evidence could be obtained for a specific tetracycline-excretion system in the resistant cells. 8. The significance of these results in relation to current theories of the antibiotic action of and resistance to the tetracycline drugs is discussed.

  12. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

    Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem

    2008-09-23

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  13. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

    Wagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-Willem

    2008-01-01

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. PMID:18796603

  14. Nucleotide excision repair in Escherichia coli.

    PubMed Central

    Van Houten, B

    1990-01-01

    One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell. PMID:2181258

  15. Expanding ester biosynthesis in Escherichia coli.

    PubMed

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2014-04-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

  16. Metabolism of Escherichia coli injured by copper.

    PubMed

    Domek, M J; Robbins, J E; Anderson, M E; McFeters, G A

    1987-01-01

    Escherichia coli injured by copper in carbonate buffer simulating the drinking water environment showed decreased oxygen utilization. Oxygraph measurements revealed that copper-injured bacteria had a rate of oxygen utilization that was less than 25% of that of control cells. Respirometry experiments measured rates over a longer period of time and showed similar trends. Nuclear magnetic resonance spectroscopy (13C nmr) and gas chromatography were used to identify differences in metabolism between healthy and injured populations of E. coli. The rate of glucose utilization by injured cells under anaerobic conditions was 64% of that of healthy cells. The rates of lactate and ethanol accumulation were 88 and 50% of the control, respectively. The 13C nmr studies of oxygenated cultures revealed differences in the accumulation of acetate and glutamine. Aerobic utilization of glucose and succinate by injured cells were 87 and 21% of the rate of the controls, respectively. Additional studies revealed injured cells had a decreased ability to reduce 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) with a variety of carbohydrate substrates. Injured cells reduced greater quantities of INT than healthy cells when NADH was used as a substrate. A comparison of metabolic end products suggested that injured cells also had considerable differences in carbon flow compared with healthy cells.

  17. Biosynthesis of ethylene glycol in Escherichia coli.

    PubMed

    Liu, Huaiwei; Ramos, Kristine Rose M; Valdehuesa, Kris Niño G; Nisola, Grace M; Lee, Won-Keun; Chung, Wook-Jin

    2013-04-01

    Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from D-xylose is reported. This route consists of four steps: D-xylose → D-xylonate → 2-dehydro-3-deoxy-D-pentonate → glycoaldehyde → EG. Respective enzymes, D-xylose dehydrogenase, D-xylonate dehydratase, 2-dehydro-3-deoxy-D-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the D-xylose → D-xylulose reaction was prevented by disrupting the D-xylose isomerase gene. The most efficient construct produced 11.7 g L(-1) of EG from 40.0 g L(-1) of D-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde → glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to D-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.

  18. Linkage map of Escherichia coli K-12, edition 8.

    PubMed Central

    Bachmann, B J

    1990-01-01

    The linkage map of Escherichia coli K-12 depicts the arrangement of genes on the circular chromosome of this organism. The basic units of the map are minutes, determined by the time-of-entry of markers from Hfr into F- strains in interrupted-conjugation experiments. The time-of-entry distances have been refined over the years by determination of the frequency of cotransduction of loci in transduction experiments utilizing bacteriophage P1, which transduces segments of DNA approximately 2 min in length. In recent years, the relative positions of many genes have been determined even more precisely by physical techniques, including the mapping of restriction fragments and the sequencing of many small regions of the chromosome. On the whole, the agreement between results obtained by genetic and physical methods has been remarkably good considering the different levels of accuracy to be expected of the methods used. There are now few regions of the map whose length is still in some doubt. In some regions, genetic experiments utilizing different mutant strains give different map distances. In other regions, the genetic markers available have not been close enough to give accurate cotransduction data. The chromosome is now known to contain several inserted elements apparently derived from lambdoid phages and other sources. The nature of the region in which the termination of replication of the chromosome occurs is now known to be much more complex than the picture given in the previous map. The present map is based upon the published literature through June of 1988. There are now 1,403 loci placed on the linkage group, which may represent between one-third and one-half of the genes in this organism. PMID:2194094

  19. Cyclomodulins in urosepsis strains of Escherichia coli.

    PubMed

    Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

    2010-06-01

    Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

  20. The extracellular RNA complement of Escherichia coli.

    PubMed

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-21

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. © 2015 The

  1. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  2. Identification of pseudouridine methyltransferase in Escherichia coli

    PubMed Central

    Ero, Rya; Peil, Lauri; Liiv, Aivar; Remme, Jaanus

    2008-01-01

    In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m3Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m3Ψ1915 is the only methylated pseudouridine in bacteria described to date. PMID:18755836

  3. Identification of pseudouridine methyltransferase in Escherichia coli.

    PubMed

    Ero, Rya; Peil, Lauri; Liiv, Aivar; Remme, Jaanus

    2008-10-01

    In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem-loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m(3)Psi) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Psi1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m(3)Psi1915 is the only methylated pseudouridine in bacteria described to date.

  4. An end-joining repair mechanism in Escherichia coli

    PubMed Central

    Chayot, Romain; Montagne, Benjamin; Mazel, Didier; Ricchetti, Miria

    2010-01-01

    Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria. PMID:20133858

  5. A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407.

    PubMed

    Crossman, Lisa C; Chaudhuri, Roy R; Beatson, Scott A; Wells, Timothy J; Desvaux, Mickael; Cunningham, Adam F; Petty, Nicola K; Mahon, Vivienne; Brinkley, Carl; Hobman, Jon L; Savarino, Stephen J; Turner, Susan M; Pallen, Mark J; Penn, Charles W; Parkhill, Julian; Turner, A Keith; Johnson, Timothy J; Thomson, Nicholas R; Smith, Stephen G J; Henderson, Ian R

    2010-11-01

    In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.

  6. Escherichia Coli Xona (Sbcb) Mutants Enhance Illegitimate Recombination

    PubMed Central

    Allgood, N. D.; Silhavy, T. J.

    1991-01-01

    Mutations of Escherichia coli K-12 were isolated that increase the frequency of deletion formation. Three of these mutations map to the gene sbcB at 43.5 min on the E. coli chromosome. Two types of mutations at sbcB have been previously defined: sbcB-type that suppress both the UV sensitivity and recombination deficiency of recBC mutants, and xonA-type that suppress only the UV sensitivity. Both types are defective for production of exonuclease I activity. The mutations isolated here were similar to xonA alleles of sbcB because they suppressed the UV sensitivity of recBC mutants but did not restore recombination proficiency. Indeed, two previously characterized xonA alleles were shown to increase the frequency of deletion formation, although an sbcB allele did not. This result demonstrates that loss of exonuclease I activity is not sufficient to confer a high deletion phenotype, rather, the product of the sbcB gene possesses some other function that is important for deletion formation. Because deletion formation in this system is recA independent and does not require extensive DNA homology, these mutations affect a pathway of illegitimate recombination. PMID:2029968

  7. Escherichia coli survival in waters: temperature dependence.

    PubMed

    Blaustein, R A; Pachepsky, Y; Hill, R L; Shelton, D R; Whelan, G

    2013-02-01

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q₁₀ model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q₁₀ equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q₁₀ equation was more accurate in rivers and coastal waters than in lakes making the value of

  8. Polymorphisms in the umuDC region of Escherichia species. [Escherichia coli; Escherichia alkalescens; Escherichia dispar; Escherichia aurescens

    SciTech Connect

    Sedgwick, S.G.; Robson, M.; Malik, F.

    1988-04-01

    The umuDC operon of Escherichia coli encodes mutagenic DNA repair. The umuDC regions of multiple isolates of E. coli, E. alkalescens, and E. dispar and a single stock of E. aurescens were mapped by nucleotide hybridization. umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long. Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract. Restriction site polymorphisms were not found around the DNA repair gene recA or polA. The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons. Possible mechanisms for the generation of these variants are discussed.

  9. Selection and characterization of beta-lactam-resistant Escherichia coli K-12 mutants.

    PubMed Central

    Jaffé, A; Chabbert, Y A; Derlot, E

    1983-01-01

    beta-Lactam-resistant mutants of Escherichia coli K-12 were selected by using 12 different beta-lactam derivatives. The mutants fell into three categories showing (i) altered permeation through reduction or loss of outer membrane porin proteins (including ompF, ompR, and envZ alleles); (ii) increase in the rate of synthesis of chromosomally mediated beta-lactamase; or (iii) defective synthesis or action of cyclic adenosine 3',5'-phosphate (cya and crp alleles). PMID:6344786

  10. Whole-Genome Sequence of Endophytic Plant Growth-Promoting Escherichia coli USML2.

    PubMed

    Tharek, Munirah; Sim, Kee-Shin; Khairuddin, Dzulaikha; Ghazali, Amir Hamzah; Najimudin, Nazalan

    2017-05-11

    Escherichia coli strain USML2 was originally isolated from the inner leaf tissues of surface-sterilized phytopathogenic-free oil palm (Elaeis guineensis Jacq.). We present here the whole-genome sequence of this plant-endophytic strain. The genome consists of a single circular chromosome of 4,502,758 bp, 4,315 predicted coding sequences, and a G+C content of 50.8%. Copyright © 2017 Tharek et al.

  11. Whole-Genome Sequence of Endophytic Plant Growth-Promoting Escherichia coli USML2

    PubMed Central

    Tharek, Munirah; Sim, Kee-Shin; Khairuddin, Dzulaikha; Najimudin, Nazalan

    2017-01-01

    ABSTRACT Escherichia coli strain USML2 was originally isolated from the inner leaf tissues of surface-sterilized phytopathogenic-free oil palm (Elaeis guineensis Jacq.). We present here the whole-genome sequence of this plant-endophytic strain. The genome consists of a single circular chromosome of 4,502,758 bp, 4,315 predicted coding sequences, and a G+C content of 50.8%. PMID:28495774

  12. RegulonDB: a database on transcriptional regulation in Escherichia coli.

    PubMed Central

    Huerta, A M; Salgado, H; Thieffry, D; Collado-Vides, J

    1998-01-01

    RegulonDB is a DataBase that integrates biological knowledge of the mechanisms that regulate the transcription initiation in Escherichia coli , as well as knowledge on the organization of the genes and regulatory signals into operons in the chromosome. The operon is the basic structure used in RegulonDB to describe the elements and properties of transcriptional regulation. The current version contains information around some 500 regulation mechanisms, essentially for sigma 70 promoters. PMID:9399800

  13. Cloning and expression in Escherichia coli of genes involved in the lysine pathway of Brevibacterium lactofermentum.

    PubMed Central

    Márquez, G; Sousa, J M; Sánchez, F

    1985-01-01

    The Brevibacterium lactofermentum genes which complement Escherichia coli lysA and asd-1 mutants were identified, respectively, as a 1.9-kilobase PstI-ClaI fragment and a 2.5-kilobase PstI fragment by cloning into pBR325. Southern blot transfers show hybridization to chromosomal fragments of identical size. The putative B. lactofermentum asd and lysA products are 44 and 48 kilodaltons, respectively. Images PMID:2864331

  14. Genome Sequence of Escherichia coli Tailed Phage Utah

    PubMed Central

    Leavitt, Justin C.; Heitkamp, Alexandra J.; Bhattacharjee, Ananda S.; Gilcrease, Eddie B.

    2017-01-01

    ABSTRACT Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah. PMID:28360173

  15. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    Sewage samples from seven locations in the United States were analyzed for Escherichia coli isolates which were resistant to trimethoprim-sulfamethoxazole (SXT). The prevalence rate of SXT resistant organisms varied between the different geographical locales. The majority of th...

  16. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    Sewage samples from seven locations in the United States were analyzed for Escherichia coli isolates which were resistant to trimethoprim-sulfamethoxazole (SXT). The prevalence rate of SXT resistant organisms varied between the different geographical locales. The majority of th...

  17. Inhibition of Thiamine Transport by Chloroethylthiamine in Escherichia coli

    PubMed Central

    Iwashima, Akio; Nose, Yoshitsugu

    1972-01-01

    Chloroethylthiamine was found to inhibit an entrapment of thiamine as thiamine monophosphate by blocking thiamine monophosphokinase in the cytoplasm after thiamine was taken up by the cells of Escherichia coli. PMID:4565550

  18. Overexpression of vsr in Escherichia coli is mutagenic.

    PubMed

    Doiron, K M; Viau, S; Koutroumanis, M; Cupples, C G

    1996-07-01

    Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.

  19. Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia.

    PubMed

    Zuo, Guanghong; Xu, Zhao; Hao, Bailin

    2013-02-01

    Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

  20. Infected hepatic Echinococcus cyst presenting as recurrent Escherichia coli empyema.

    PubMed

    Chang, R; Higgins, M; DiLisio, R; Hawasli, A; Camaro, L G; Khatib, R

    1993-03-01

    An 81-year-old man, previously a shepherd in Italy, presented with recurrent Escherichia coli empyema over an 8-month period. His empyema was caused by an infected, nonviable hepatic Echinococcus cyst that eroded the diaphragm and led to intermittent spillage and pleural seeding. This case demonstrates that when dealing with Escherichia coli empyema, a subdiaphragmatic source ought to be suspected, and among immigrants from areas with prevalent hydatid disease, infected hepatic Echinococcus cyst might rarely be the cause.

  1. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

    PubMed

    FREIFELDER, D; MAALOE, O

    1964-10-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

  2. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI

    PubMed Central

    Freifelder, David; Maaløe, Ole

    1964-01-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987–990. 1964.—Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process. PMID:14219063

  3. Comparative sequence analysis of enteroaggregative Escherichia coli heat-stable enterotoxin 1 identified in Korean and Japanese Escherichia coli strains.

    PubMed

    Seo, Dong Joo; Choi, SunKeum; Jeon, Su Been; Jeong, Suntak; Park, Hyunkyung; Lee, Bog-Hieu; Kim, Geun-Bae; Yang, Soo-Jin; Nishikawa, Yoshikazu; Choi, Changsun

    2017-02-21

    The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1.

  4. The Escherichia coli Peripheral Inner Membrane Proteome*

    PubMed Central

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

    2013-01-01

    Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with

  5. Microdiesel: Escherichia coli engineered for fuel production.

    PubMed

    Kalscheuer, Rainer; Stölting, Torsten; Steinbüchel, Alexander

    2006-09-01

    Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l(-1) and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

  6. Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

    PubMed Central

    Gilbert, J V; Plaut, A G; Fishman, Y; Wright, A

    1988-01-01

    We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region. Images PMID:3294181

  7. Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2016

    DTIC Science & Technology

    2017-06-30

    women.5 Screening practices may also contribute to higher rates of E. coli infections among females of reproductive age, as the Infectious Disease...Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS...and prevalence among all beneficiaries seeking care within the Military Health System (MHS). This report describes demographics, clinical

  8. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Escherichia coli serological reagents. 866.3255 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255 Escherichia...

  9. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255 Escherichia...

  10. Biocontrol of Escherichia coli O157

    PubMed Central

    Boyacioglu, Olcay; Sharma, Manan; Sulakvelidze, Alexander; Goktepe, Ipek

    2013-01-01

    The effect of a bacteriophage cocktail (EcoShield™) that is specific against Escherichia coli O157:H7 was evaluated against a nalidixic acid-resistant enterohemorrhagic E. coli O157:H7 RM4407 (EHEC) strain on leafy greens stored under either (1) ambient air or (2) modified atmosphere (MA; 5% O2/35% CO2/60% N2). Pieces (~2 × 2 cm2) of leafy greens (lettuce and spinach) inoculated with 4.5 log CFU/cm2 EHEC were sprayed with EcoShield™ (6.5 log PFU/cm2). Samples were stored at 4 or 10°C for up to 15 d. On spinach, the level of EHEC declined by 2.38 and 2.49 log CFU/cm2 at 4 and 10°C, respectively, 30 min after phage application (p ≤ 0.05). EcoShield™ was also effective in reducing EHEC on the surface of green leaf lettuce stored at 4°C by 2.49 and 3.28 log units in 30 min and 2 h, respectively (p ≤ 0.05). At 4°C under atmospheric air, the phage cocktail significantly (p ≤ 0.05) lowered the EHEC counts in one day by 1.19, 3.21 and 3.25 log CFU/cm2 on spinach, green leaf and romaine lettuce, respectively compared with control (no bacteriophage) treatments. When stored under MA at 4°C, phages reduced (p ≤ 0.05) EHEC populations by 2.18, 3.50 and 3.13 log CFU/cm2, on spinach, green leaf and romaine lettuce. At 10°C, EHEC reductions under atmospheric air storage were 1.99, 3.90 and 3.99 log CFU/cm2 (p ≤ 0.05), while population reductions under MA were 3.08, 3.89 and 4.34 logs on spinach, green leaf and romaine lettuce, respectively, compared with controls (p ≤ 0.05). The results of this study showed that bacteriophages were effective in reducing the levels of E. coli O157:H7 on fresh leafy produce, and that the reduction was further improved when produce was stored under the MA conditions. PMID:23819107

  11. [Production of coenzyme Q10 by metabolically engineered Escherichia coli].

    PubMed

    Dai, Guanping; Miao, Liangtian; Sun, Tao; Li, Qingyan; Xiao, Dongguang; Zhang, Xueli

    2015-02-01

    Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.

  12. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  13. Shiga toxin-producing Escherichia coli

    PubMed Central

    Etcheverría, Analía Inés; Padola, Nora Lía

    2013-01-01

    Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Outbreaks are linked to bovine food sources. STEC O157:H7 has been responsible for the most severe outbreaks worldwide. However, non-O157 serotypes have emerged as important enteric pathogens in several countries. The main virulence factor of STEC is the production of Shiga toxins 1 and 2. Additional virulence markers are a plasmid-encoded enterohemolysin (ehxA), an autoagglutinating adhesin (Saa), a catalase-peroxidase (katP), an extracellular serine protease (espP), a zinc metalloprotease (stcE), a subtilase cytotoxin (subAB), among others. Other virulence factors are intimin and adhesins that had a roll in the adherence of STEC to bovine colon. This review focuses on the virulence traits of STEC and especially on those related to the adhesion to bovine colon. The known of the interaction between STEC and the bovine host is crucial to develop strategies to control cattle colonization. PMID:23624795

  14. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  15. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1986-03-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. Focus is on the two final steps in alcohol synthesis, which are catalyzed by alcohol dehydrogenase and acetaldehyde CoA dehydrogenase. We have isolated a series of mutations affecting the expression of these enzymes. Some of these mutations are in the structural genes for these enzymes; others affect the regulation of the adh operon. We have recently cloned the genes coding for these enzymes and are now studying the effect of multiple copies of the adh gene on fermentative growth and its regulation. A recently invented technique, proton suicide has allowed the selection of a variety of novel mutants affecting fermentation which are presently being characterized. We have isolated a comprehensive collection of operon fusions in which the lacZ structural gene is fused to promoters that are inactive aerobically but active anaerobically. Although these genes (like adh) are only expressed under anaerobic conditions, the level of induction varies from two-fold to nearly 100-fold. The nitrogen source, medium pH, nature of the buffer, presence of alternative electron acceptors (e.g., nitrate), and other factors exert a great effect on the expression of many of these genes. In the near future we will investigate control mechanisms common to the adh operon and other anaerobically regulated genes.

  16. Regulation of Glutamine Transport in Escherichia coli.

    PubMed Central

    Willis, R C; Iwata, K K; Furlong, C E

    1975-01-01

    The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate. PMID:238938

  17. ESCHERICHIA COLI Gene Induction by Alkylation Treatment

    PubMed Central

    Volkert, Michael R.; Nguyen, Dinh C.; Beard, K. Christopher

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased β-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes. PMID:3080354

  18. Escherichia coli gene induction by alkylation treatment.

    PubMed

    Volkert, M R; Nguyen, D C; Beard, K C

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.

  19. Antimicrobial-resistant Invasive Escherichia coli, Spain

    PubMed Central

    Oteo, Jesús; Lázaro, Edurne; de Abajo, Francisco J.; Baquero, Fernando; Campos, José

    2005-01-01

    To address the public health problem of antimicrobial resistance, the European Union founded the European Antimicrobial Resistance Surveillance System. A network of 32 Spanish hospitals, serving ≈9.6 million persons, submitted antimicrobial-susceptibility data on 7,098 invasive Escherichia coli species (2001–2003). Resistance to ampicillin, cotrimoxazole, ciprofloxacin, gentamicin, and tobramycin was found at rates of 59.9%, 32.6%, 19.3%, 6.8%, and 5.3%, respectively. Resistance to multiple drugs increased from 13.8% in 2001 to 20.6% in 2003 (p <0.0001). Antimicrobial consumption data were obtained from the Spanish National Health System. In spite of decreased cephalosporin and β-lactam use, overall extended-spectrum β-lactamase production increased from 1.6% (2001) to 4.1% (2003) (p <0.0001), mainly due to the rising prevalence of cefotaximases. Resistance to ciprofloxacin significantly increased, mostly in community-onset infections, which coincided with a rise in community quinolone use. Cotrimoxazole resistance remained stable at ≈30%, even though its use was dramatically reduced. PMID:15829192

  20. Ribonuclease Sensitivity of Escherichia coli Ribosomes

    PubMed Central

    Santer, Melvin; Smith, Josephine R.

    1966-01-01

    Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099–1110. 1966.—The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10−4m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities. Images PMID:5332866

  1. Genotoxicity of Graphene in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Sharma, Ananya

    Rapid advances in nanotechnology necessitate assessment of the safety of nanomaterials in the resulting products and applications. One key nanomaterial attracting much interest in many areas of science and technology is graphene. Graphene is a one atom thick carbon allotrope arranged in a two-dimensional honeycomb lattice. In addition to being extremely thin, graphene has several extraordinary physical properties such as its exceptional mechanical strength, thermal stability, and high electrical conductivity. Graphene itself is relatively chemically inert and therefore pristine graphene must undergo a process called functionalization, which is combination of chemical and physical treatments that change the properties of graphene, to make it chemically active. Functionalization of graphene is of crucial importance as the end application of graphene depends on proper functionalization. In the field of medicine, graphene is currently a nanomaterial of high interest for building biosensors, DNA transistors, and probes for cancer detection. Despite the promising applications of graphene in several areas of biomedicine, there have been only few studies in recent years that focus on evaluating cytotoxicity of graphene on cells, and almost no studies that investigate how graphene exposure affects cellular genetic material. Therefore, in this study we used a novel approach to evaluate the genotoxicity, i.e., the effects of graphene on DNA, using Escherichia coli as a prokaryotic model organism.

  2. The DNA exonucleases of Escherichia coli

    PubMed Central

    Lovett, Susan T.

    2014-01-01

    DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB) and III (dnaQ/mutD), Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG) and X (exoX), the RecBCD, RecJ, and RecE exonucleases, SbcCD endo/exonuclease, the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo) and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life. PMID:26442508

  3. Type II toxin-antitoxin systems are unevenly distributed among Escherichia coli phylogroups.

    PubMed

    Fiedoruk, Krzysztof; Daniluk, Tamara; Swiecicka, Izabela; Sciepuk, Malgorzata; Leszczynska, Katarzyna

    2015-01-01

    Type II toxin-antitoxin systems (TAs) are bicistronic operons ubiquitous in prokaryotic genomes, displaying multilevel association with cell physiology. Various possible functions have been assigned to TAs, ranging from beneficial for their hosts, such as a stress response, dormancy and protection against genomic parasites, to detrimental or useless functions, such as selfish alleles. As there is a link between several Escherichia coli features (e.g. virulence, lifestyle) and the phylogeny of this species, we hypothesized a similar association with TAs. Using PCR we studied the distribution of 15 chromosomal and plasmidic type II TA loci in 84 clinical E. coli isolates in relation to their main phylogenetic groups (A, B1, B2 and D). In addition, we performed in silico searching of these TA loci in 60 completely sequenced E. coli genomes deposited in GenBank. The highest number of TA loci per strain was observed in group A (mean 8.2, range 5-12) and the lowest in group B2 (mean 4.2, range 2-8). Moreover, significant differences in the prevalence of nine chromosomal TAs among E. coli phylogroups were noted. In conclusion, the presence of some chromosomal TAs in E. coli is phylogroup-related rather than a universal feature of the species. In addition, their limited collection in group B2 clearly distinguish it from the other E. coli phylogroups.

  4. Enterotoxigenic Escherichia coli Multilocus Sequence Types in Guatemala and Mexico

    PubMed Central

    Klena, John; Rodas, Claudia; Bourgeois, August Louis; Torres, Olga; Svennerholm, Ann-Mari; Sjöling, Åsa

    2010-01-01

    The genetic backgrounds of 24 enterotoxigenic Escherichia coli (ETEC) strains from Mexico and Guatemala expressing heat-stable toxin (ST) and coli surface antigen 6 (CS6) were analyzed. US travelers to these countries and resident children in Guatemala were infected by ETEC strains of sequence type 398, expressing STp and carrying genetically identical CS6 sequences. PMID:20031063

  5. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses

    USDA-ARS?s Scientific Manuscript database

    Enterohemorrhagic E. coli (EHEC) are Shiga toxin–producing Escherichia coli (STEC) associated with the most severe forms of foodborne illnesses. The United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) has identified a higher percentage of non-O157 EHEC compared to E....

  6. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution.

    PubMed

    von Mentzer, Astrid; Connor, Thomas R; Wieler, Lothar H; Semmler, Torsten; Iguchi, Atsushi; Thomson, Nicholas R; Rasko, David A; Joffre, Enrique; Corander, Jukka; Pickard, Derek; Wiklund, Gudrun; Svennerholm, Ann-Mari; Sjöling, Åsa; Dougan, Gordon

    2014-12-01

    Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea, produce heat-stable and/or heat-labile enterotoxins and at least 25 different colonization factors that target the intestinal mucosa. The genes encoding the enterotoxins and most of the colonization factors are located on plasmids found across diverse E. coli serogroups. Whole-genome sequencing of a representative collection of ETEC isolated between 1980 and 2011 identified globally distributed lineages characterized by distinct colonization factor and enterotoxin profiles. Contrary to current notions, these relatively recently emerged lineages might harbor chromosome and plasmid combinations that optimize fitness and transmissibility. These data have implications for understanding, tracking and possibly preventing ETEC disease.

  7. Escherichia coli Pathotypes Occupy Distinct Niches in the Mouse Intestine

    PubMed Central

    Meador, Jessica P.; Caldwell, Matthew E.; Cohen, Paul S.

    2014-01-01

    Since the first step of the infection process is colonization of the host, it is important to understand how Escherichia coli pathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strain E. coli EDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains how E. coli EDL933 overcomes colonization resistance imparted by some, but not all, commensal E. coli strains. Here we sought to determine if other E. coli pathogens use a similar strategy. We found that uropathogenic E. coli CFT073 and enteropathogenic E. coli E2348/69 occupy intestinal niches that are distinct from that of E. coli EDL933. In contrast, two enterohemorrhagic strains, E. coli EDL933 and E. coli Sakai, occupy the same niche, suggesting that strategies to prevent colonization by a given pathotype should be effective against other strains of the same pathotype. However, we found that a combination of commensal E. coli strains that can prevent colonization by E. coli EDL933 did not prevent colonization by E. coli CFT073 or E. coli E2348/69. Our results indicate that development of probiotics to target multiple E. coli pathotypes will be problematic, as the factors that govern niche occupation and hence stable colonization vary significantly among strains. PMID:24566621

  8. Draft Genome Sequence of Uropathogenic Escherichia coli Strain NB8

    PubMed Central

    Mi, Zu-huang; Wang, Chun-xin; Zhu, Jian-ming

    2016-01-01

    Escherichia coli NB8 is a clinical pyelonephritis isolate. Here, we report the draft genome sequence of uropathogenic E. coli NB8, which contains drug resistance genes encoding resistance to beta-lactams, aminoglycosides, quinolones, macrolides, colistin, sulfonamide-trimethoprim, and tetracycline. NB8 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis. PMID:27609920

  9. Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

    PubMed

    Lindsey, Rebecca L; Garcia-Toledo, L; Fasulo, D; Gladney, L M; Strockbine, N

    2017-09-01

    Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies. Published by Elsevier B.V.

  10. Soil solarization reduces Escherichia coli O157:H7 and total Escherichia coli on cattle feedlot pen surfaces

    USDA-ARS?s Scientific Manuscript database

    Feedlot pen soils are a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM)....

  11. PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites

    PubMed Central

    Hershberg, Ruti; Bejerano, Gill; Santos-Zavaleta, Alberto; Margalit, Hanah

    2001-01-01

    PromEC is an updated compilation of Escherichia coli mRNA promoter sequences. It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E.coli chromosome, as well as the actual sequences in the promoter region. The database was updated as of July 2000 and includes 472 entries. PromEC is accessible at http://bioinfo.md.huji.ac.il/marg/promec PMID:11125111

  12. Urinary Tract Physiological Conditions Promote Ciprofloxacin Resistance in Low-Level-Quinolone-Resistant Escherichia coli.

    PubMed

    Martín-Gutiérrez, Guillermo; Rodríguez-Beltrán, Jerónimo; Rodríguez-Martínez, José Manuel; Costas, Coloma; Aznar, Javier; Pascual, Álvaro; Blázquez, Jesús

    2016-07-01

    Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs.

  13. Urinary Tract Physiological Conditions Promote Ciprofloxacin Resistance in Low-Level-Quinolone-Resistant Escherichia coli

    PubMed Central

    Rodríguez-Martínez, José Manuel; Costas, Coloma; Aznar, Javier; Pascual, Álvaro

    2016-01-01

    Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli. However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli. Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs. PMID:27139482

  14. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  15. Integrated Genomic Map from Uropathogenic Escherichia coli J96

    PubMed Central

    Melkerson-Watson, Lyla J.; Rode, Christopher K.; Zhang, Lixin; Foxman, Betsy; Bloch, Craig A.

    2000-01-01

    Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratory E. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10 insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis in NotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of

  16. The Melibiose Transporter of Escherichia coli

    PubMed Central

    Fuerst, Oliver; Lin, Yibin; Granell, Meritxell; Leblanc, Gérard; Padrós, Esteve; Lórenz-Fonfría, Víctor A.; Cladera, Josep

    2015-01-01

    We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na+-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na+ and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200–330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na+ ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na+ ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na+ binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na+ binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions. PMID:25971963

  17. Polyamine transport inEscherichia coli.

    PubMed

    Igarashi, K; Kashiwagi, K

    1996-03-01

    The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) inEscherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were periplasmic transport systems consisting of four kinds of proteins: pPT104 clone encoded potA, -B,-C, and -D proteins and pPT79 clone encoded potF, -G, -H, and -I proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones. PotD and -F proteins were periplasmic substrate binding proteins and potA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and potH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of potA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (potE protein) haveing twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine.

  18. Novel Mechanism of Escherichia coli Porin Regulation

    PubMed Central

    Castillo-Keller, Maria; Vuong, Phu; Misra, Rajeev

    2006-01-01

    A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3′-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC—a remnant porin gene from the cryptic phage qsr′ (DLP12) genome—and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3′ end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq. PMID:16385048

  19. Mutational Consequences of Ciprofloxacin in Escherichia coli.

    PubMed

    Song, Lisa Yun; Goff, Marisa; Davidian, Christina; Mao, Zhiyuan; London, Marisa; Lam, Karen; Yung, Madeline; Miller, Jeffrey H

    2016-10-01

    We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Serogroups of Escherichia coli from drinking water.

    PubMed

    Ramteke, P W; Tewari, Suman

    2007-07-01

    Fifty seven isolates of thermotolerant E. coli were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E. coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shiga-toxin producing E. coli, STEC). All the pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and cotrimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied. Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As(3+) followed by Cr(6+) were transferred more frequently.

  1. Control of Acid Resistance in Escherichia coli

    PubMed Central

    Castanie-Cornet, Marie-Pierre; Penfound, Thomas A.; Smith, Dean; Elliott, John F.; Foster, John W.

    1999-01-01

    Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor ςS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ςS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ςS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the

  2. Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells

    PubMed Central

    Cuevas-Ramos, Gabriel; Petit, Claude R.; Marcq, Ingrid; Boury, Michèle; Oswald, Eric; Nougayrède, Jean-Philippe

    2010-01-01

    Escherichia coli is a normal inhabitant of the human gut. However, E. coli strains of phylogenetic group B2 harbor a genomic island called “pks” that codes for the production of a polyketide-peptide genotoxin, Colibactin. Here we report that in vivo infection with E. coli harboring the pks island, but not with a pks isogenic mutant, induced the formation of phosphorylated H2AX foci in mouse enterocytes. We show that a single, short exposure of cultured mammalian epithelial cells to live pks+ E. coli at low infectious doses induced a transient DNA damage response followed by cell division with signs of incomplete DNA repair, leading to anaphase bridges and chromosome aberrations. Micronuclei, aneuploidy, ring chromosomes, and anaphase bridges persisted in dividing cells up to 21 d after infection, indicating occurrence of breakage–fusion–bridge cycles and chromosomal instability. Exposed cells exhibited a significant increase in gene mutation frequency and anchorage-independent colony formation, demonstrating the infection mutagenic and transforming potential. Therefore, colon colonization with these E. coli strains harboring the pks island could contribute to the development of sporadic colorectal cancer. PMID:20534522

  3. Environmental Escherichia coli: Ecology and public health implications - A review

    USGS Publications Warehouse

    Jang, Jeonghwan; Hur, Hor-Gil; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Yan, Tao; Ishii, Satoshi

    2017-01-01

    Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

  4. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development.

    PubMed

    Yang, Shih-Chun; Lin, Chih-Hung; Aljuffali, Ibrahim A; Fang, Jia-You

    2017-08-01

    Food contamination by pathogenic microorganisms has been a serious public health problem and a cause of huge economic losses worldwide. Foodborne pathogenic Escherichia coli (E. coli) contamination, such as that with E. coli O157 and O104, is very common, even in developed countries. Bacterial contamination may occur during any of the steps in the farm-to-table continuum from environmental, animal, or human sources and cause foodborne illness. To understand the causes of the foodborne outbreaks by E. coli and food-contamination prevention measures, we collected and investigated the past 10 years' worldwide reports of foodborne E. coli contamination cases. In the first half of this review article, we introduce the infection and symptoms of five major foodborne diarrheagenic E. coli pathotypes: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli/enterohemorrhagic E. coli (STEC/EHEC), Shigella/enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and enterotoxigenic E. coli (ETEC). In the second half of this review article, we introduce the foodborne outbreak cases caused by E. coli in natural foods and food products. Finally, we discuss current developments that can be applied to control and prevent bacterial food contamination.

  5. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

    E . coli diarrheal disease in man and domestic animals. Fundamentally, the design of the vaccine is based on the well- documented ability of cholera antitoxin to neutralize both cholera and heat- labile E . coli enterotoxins and on the ability of certain E . coli antigens to enhance the immune response to cholera toxoid and possibly whole-cell Cholera Vaccine, as

  6. Genetic method to analyze essential genes of Escherichia coli.

    PubMed

    Hupert-Kocurek, Katarzyna; Sage, Jay M; Makowska-Grzyska, Magdalena; Kaguni, Jon M

    2007-11-01

    The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.

  7. Anaerobically expressed Escherichia coli genes identified by operon fusion techniques.

    PubMed Central

    Choe, M; Reznikoff, W S

    1991-01-01

    Genes that are expressed under anaerobic conditions were identified by operon fusion techniques with a hybrid bacteriophage of lambda and Mu, lambda placMu53, which creates transcriptional fusions to lacZY. Cells were screened for anaerobic expression on XG medium. Nine strains were selected, and the insertion point of the hybrid phage in each strain was mapped on the Escherichia coli chromosome linkage map. The anaerobic and aerobic expression levels of these genes were measured by beta-galactosidase assays in different medium conditions and in the presence of three regulatory mutations (fnr, narL, and rpoN). The anaerobically expressed genes (aeg) located at minute 99 (aeg-99) and 75 (aeg-75) appeared to be partially regulated by fnr, and aeg-93 is tightly regulated by fnr. aeg-60 requires a functional rpoN gene for its anaerobic expression. aeg-46.5 is repressed by narL. aeg-65A and aeg-65C are partially controlled by fnr but only in media containing nitrate or fumarate. aeg-47.5 and aeg-48.5 were found to be anaerobically induced only in rich media. The effects of a narL mutation on aeg-46.5 expression were observed in all medium conditions regardless of the presence or absence of nitrate. This suggests that narL has a regulatory function in the absence of exogenously added nitrate. PMID:1917846

  8. Inhibiting translation elongation can aid genome duplication in Escherichia coli.

    PubMed

    Myka, Kamila K; Hawkins, Michelle; Syeda, Aisha H; Gupta, Milind K; Meharg, Caroline; Dillingham, Mark S; Savery, Nigel J; Lloyd, Robert G; McGlynn, Peter

    2016-12-11

    Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

  9. Rapid Sterilization of Escherichia coli by Solution Plasma Process

    NASA Astrophysics Data System (ADS)

    Andreeva, Nina; Ishizaki, Takahiro; Baroch, Pavel; Saito, Nagahiro

    2012-12-01

    Solution plasma (SP), which is a discharge in the liquid phase, has the potential for rapid sterilization of water without chemical agents. The discharge showed a strong sterilization performance against Escherichia coli bacteria. The decimal value (D value) of the reduction time for E. coli by this system with an electrode distance of 1.0 mm was estimated to be approximately 1.0 min. Our discharge system in the liquid phase caused no physical damage to the E. coli and only a small increase in the temperature of the aqueous solution. The UV light generated by the discharge was an important factor in the sterilization of E. coli.

  10. Human Meningitis-Associated Escherichia coli

    PubMed Central

    KIM, KWANG SIK

    2016-01-01

    E. coli is the most common Gram-negative bacillary organism causing meningitis and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis contributes to such mortality and morbidity. Recent reports of E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. Studies using in vitro and in vivo models of the blood-brain barrier have shown that E. coli meningitis follows a high-degree of bacteremia and invasion of the blood-brain barrier. E. coli invasion of the blood-brain barrier, the essentials step in the development of E. coli meningitis, requires specific microbial and host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors contributing to E. coli invasion of the blood-brain barrier is shown to be efficient in preventing E. coli penetration into the brain. The basis for requiring a high-degree of bacteremia for E. coli penetration of the blood-brain barrier, however, remains unclear. Continued investigation on the microbial and host factors contributing to a high-degree of bacteremia and E. coli invasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis. PMID:27223820

  11. [Frequency, risk factors and vaginal colonization due to Escherichia coli].

    PubMed

    González Pedraza Avilés, Alberto; Sánchez Hernández, Gabriela; Ponce Rosas, Raúl Efrén

    2004-02-01

    Recent studies associate Escherichia coli with symptomatic infections at vaginal level, mainly associated to changes in the normal flora taken place by a series of factors characteristic of the host. To recognize their colonization frequency and these factors, it becomes important due to their association with perinatal complications, besides considering this colonization like the critical step preceding urinary tract infection. To determine the frequency of colonization of Escherichia coli in 519 female patients, the role of the bacterium in the vaginal ecology likes probable cause of clinical manifestations and to recognize the associate's factors of risk with its vaginal colonization. 519 women were studied: 350 symptomatic and 169 asymptomatic. Vaginal swab specimens were inoculated onto the routine mediums. Associations of Escherichia coli with various risk factors were examined by using odds ratios (ORs) and 95% confidence intervals, and statistical significance was assessed by the Chi statistic or Fischer's exact test. Overall Escherichia coli was isolated from 95 (18.3%) of the women. Factors that were significantly associated with vaginal carriage of E. coli were the age extreme groups, the climacteric, and the bad genital habits. The highest frequency of vaginal colonization for Escherichia coli was presented in the population groups where there is hormonal deficiency, mainly of estrogens of the type estradiol. The vaginal colonization for E. coli doesn't associate to sexual behavior. Although E. coli doesn't produce defined symptoms at vaginal level, the relatively low carriage rate indicates that this organism should not be considered as part of the normal indigenous vaginal flora and that it should take into account due to the perinatal complication it is associated.

  12. The Biology of the Escherichia coli Extracellular Matrix

    PubMed Central

    Hufnagel, David A.; DePas, William H.; Chapman, Matthew R.

    2015-01-01

    Chapter Summary Escherichia coli (E. coli) is one of the world’s best-characterized organisms, as it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere; in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix, primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces, and resistance to desiccation, the host immune system and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this book chapter, which concludes with insights into the future of E. coli biofilm research and potential therapies. PMID:26185090

  13. Sonodynamic action of curcumin on foodborne bacteria Bacillus cereus and Escherichia coli.

    PubMed

    Wang, Xinna; Ip, Margaret; Leung, Albert Wingnang; Yang, Zhengrong; Wang, Pan; Zhang, Baoting; Ip, Siupo; Xu, Chuanshan

    2015-09-01

    Bacterial contamination is an important cause of foodborne diseases. The present study aimed to investigate sonodynamic action of curcumin on foodborne bacteria Bacillus cereus (B. cereus) and Escherichia coli (E. coli). The uptake of curcumin was measured for optimizing the concentration incubation time before ultrasound sonication, and colony forming units (CFU) were counted after ultrasound treatment. The chromosomal DNA fragmentation of bacteria was analyzed and the effect of hypoxic condition on the antibacterial efficacy of sonodynamic action of curcumin was also assessed in this study. The results showed that the maximum uptake of curcumin in B. cereus and E. coli occurred in 50min after curcumin incubation. Curcumin had sonodynamic bactericidal activity in a curcumin dose-dependent manner, and 5.6-log reduction in CFU of B. cereus was observed after curcumin treatment (2.0μM), however, only 2-log reduction in CFU of E. coli after 40μM curcumin treatment. No significant change in chromosomal DNA was found after the combined treatment of curcumin and ultrasound. The survival of B. cereus and E. coli after sonodynamic treatment in hypoxic group was significantly higher than that in normal oxygen group. These findings indicated that sonodynamic action of curcumin had significant inactivation effect on foodborne bacteria, and B. cereus was more sensitive to sonodynamic treatment of curcumin than E. coli. Sonodynamic antibacterial activity of curcumin might be dependent on the oxygen environment.

  14. Infection by verocytotoxin-producing Escherichia coli.

    PubMed Central

    Karmali, M A

    1989-01-01

    Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated

  15. Plasmid Diversity and Adaptation Analyzed by Massive Sequencing of Escherichia coli Plasmids.

    PubMed

    de Toro, María; Garcilláon-Barcia, M Pilar; De La Cruz, Fernando

    2014-12-01

    Whole-genome sequencing is revolutionizing the analysis of bacterial genomes. It leads to a massive increase in the amount of available data to be analyzed. Bacterial genomes are usually composed of one main chromosome and a number of accessory chromosomes, called plasmids. A recently developed methodology called PLACNET (for plasmid constellation networks) allows the reconstruction of the plasmids of a given genome. Thus, it opens an avenue for plasmidome analysis on a global scale. This work reviews our knowledge of the genetic determinants for plasmid propagation (conjugation and related functions), their diversity, and their prevalence in the variety of plasmids found by whole-genome sequencing. It focuses on the results obtained from a collection of 255 Escherichia coli plasmids reconstructed by PLACNET. The plasmids found in E. coli represent a nonaleatory subset of the plasmids found in proteobacteria. Potential reasons for the prevalence of some specific plasmid groups will be discussed and, more importantly, additional questions will be posed.

  16. Intestinal Colonization by Enterotoxigenic Escherichia coli.

    DTIC Science & Technology

    1980-09-01

    E . coli is mediated by specific types of pili. These pili are antigenic and can be used in diagnosing enterotoxigenic E . coli infections. They are also good protective antigens. When pregnant dams are vaccinated parenterally or orally with pili on live piliated bacteria, they secrete antibodies against the pili in their milk. Neonates suckling dams so vaccinated are passively protected against fatal challenge by enterotoxigenic E . coli . Pili are also good candidate protective antigens for the development of vaccines to protect by

  17. [Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli].

    PubMed

    Ptitsyn, L R; Fatova, M A; Stepanov, A I

    1990-02-01

    Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

  18. Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli

    PubMed Central

    Commereuc, Morgane; Weill, Francois-Xavier; Loukiadis, Estelle; Gouali, Malika; Gleizal, Audrey; Kormann, Raphaël; Ridel, Christophe; Frémeaux-Bacchi, Véronique; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Abstract A widespread belief is that typical hemolytic and uremic syndrome (HUS) does not recur. We report the case of a patient infected twice with raw milk taken from his own cow and containing a Shiga toxin–producing Escherichia coli O174:H21 that induced recurrent HUS causing severe renal and cerebral disorders. A genomic comparison of the human and bovine Shiga toxin–producing Escherichia coli O174:H21 isolates revealed that they were identical. Typical HUS may recur. Since milk from this animal was occasionally distributed locally, thereby posing a serious threat for the whole village, this particular cow was destroyed. PMID:26735524

  19. Diarrheagenic Escherichia coli in Children from Costa Rica

    PubMed Central

    Pérez, Cristian; Gómez-Duarte, Oscar G.; Arias, María L.

    2010-01-01

    More than 5,000 diarrheal cases per year receive medical care at the National Children's Hospital of Costa Rica, and nearly 5% of them require hospitalization. A total of 173 Escherichia coli strains isolated from children with diarrhea were characterized at the molecular, serologic, and phenotypic level. Multiplex and duplex polymerase chain reactions were used to detect the six categories of diarrheagenic E. coli. Thirty percent (n = 52) of the strains were positive, indicating a high prevalence among the pediatric population. Enteropathogenic E. coli and enteroinvasive E. coli pathotypes were the most prevalent (21% and 19%, respectively). Pathogenic strains were distributed among the four E. coli phylogenetic groups A, B1, B2, and D, with groups A and B1 the most commonly found. This study used molecular typing to evaluate the prevalence of diarrheagenic E. coli reported in Costa Rica and demonstrated the importance of these pathotypes in the pediatric population. PMID:20682870

  20. Genes and proteins of Escherichia coli K-12.

    PubMed

    Riley, M

    1998-01-01

    GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins, representing groups of paralogous genes, with PAM values, percent identity of amino acids, length of alignment and percent aligned. GenProtEC can be accessed at the URL http://www.mbl.edu/html/ecoli.html

  1. Large Surface Blebs on Escherichia coli Heated to Inactivating Temperatures

    PubMed Central

    Scheie, Paul; Ehrenspeck, Susan

    1973-01-01

    Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and Bs-1 heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event. Images PMID:4196258

  2. Expression of staphylococcal enterotoxin C1 in Escherichia coli.

    PubMed Central

    Bohach, G A; Schlievert, P M

    1987-01-01

    The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active. Images PMID:3542834

  3. Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin

    PubMed Central

    2011-01-01

    Background The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin. Results Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw). Conclusions By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were

  4. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    PubMed Central

    2009-01-01

    Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead to unwanted secondary

  5. Multidrug-resistant Escherichia coli in Asia: epidemiology and management.

    PubMed

    Sidjabat, Hanna E; Paterson, David L

    2015-05-01

    Escherichia coli has become multiresistant by way of production of a variety of β-lactamases. The prevalence of CTX-M-producing E. coli has reached 60-79% in certain parts of Asia. The acquisition of CTX-M plasmids by E. coli sequence type 131, a successful clone of E. coli, has caused further dissemination of CTX-M-producing E. coli. The prevalence of carbapenemase-producing E. coli, especially Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase (NDM)-producing E. coli has been increasing in Asia. K. pneumoniae carbapenemase and NDM have now been found in E. coli sequence type 131. The occurrence of NDM-producing E. coli is a major concern particularly in the Indian subcontinent, but now elsewhere in Asia as well. There are multiple reasons why antibiotic resistance in E. coli in Asia has reached such extreme levels. Approaches beyond antibiotic therapy, such as prevention of antibiotic resistance by antibiotic stewardship and protecting natural microbiome, are strategies to avoid further spread of antibiotic resistance.

  6. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

    PubMed Central

    Jameson, Katie H.; Wilkinson, Anthony J.

    2017-01-01

    Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis. PMID:28075389

  7. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli.

    PubMed

    Jameson, Katie H; Wilkinson, Anthony J

    2017-01-10

    Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis.

  8. Characterization of multi-antibiotic-resistant Escherichia coli Isolated from beef cattle in Japan.

    PubMed

    Yamamoto, Shiori; Nakano, Motoki; Kitagawa, Wataru; Tanaka, Michiko; Sone, Teruo; Hirai, Katsuya; Asano, Kozo

    2014-01-01

    The emergence of multiple-antibiotic-resistance bacteria is increasing, which is a particular concern on livestock farms. We previously isolated 1,347 antimicrobial-resistant (AMR) Escherichia coli strains from the feces of beef cattle on 14 Japanese farms. In the present study, the genetic backgrounds and phylogenetic relationships of 45 AMR isolates were characterized by the chromosome phylotype, AMR phenotype, AMR genotype, and plasmid type. These isolates were classified into five chromosome phylotypes, which were closely linked to the farms from which they were isolated, suggesting that each farm had its own E. coli phylotype. AMR phenotype and plasmid type analyses yielded 8 and 14 types, all of which were associated with the chromosomal phylotype and, thus, to the original farms. AMR genotype analysis revealed more variety, with 16 types, indicating both inter- and intra-farm diversity. Different phylotype isolates from the same farm shared highly similar plasmid types, which indicated that plasmids with AMR genes could be transferred between phylotypes, thereby generating multi-antibiotic-resistant microorganisms. This ecological study demonstrated that the chromosome phylotype was strongly correlated with the farm from which they were isolated, while the AMR phenotype, genotype, and plasmid type were generally correlated with the chromosome phylotype and farm source.

  9. Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species.

    PubMed

    Potter, S; Yang, X; Boulanger, M J; Ishiguro, E E

    1998-04-01

    The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.

  10. Chromosomal location of the fosA3 and blaCTX-M genes in Proteus mirabilis and clonal spread of Escherichia coli ST117 carrying fosA3-positive IncHI2/ST3 or F2:A-:B- plasmids in a chicken farm.

    PubMed

    He, Dandan; Liu, Lanping; Guo, Baowei; Wu, Shengjun; Chen, Xiaojie; Wang, Jing; Zeng, Zhenling; Liu, Jian-Hua

    2017-04-01

    The aim of this study was to investigate the spread and location of the fosA3 gene among Enterobacteriaceae from diseased broiler chickens. Twenty-nine Escherichia coli and seven Proteus mirabilis isolates recovered from one chicken farm were screened for the presence of plasmid-mediated fosfomycin resistance genes by PCR. The clonal relatedness of fosA3-positive isolates, the transferability and location of fosA3, and the genetic context of the fosA3 gene were determined. Seven P. mirabilis isolates with three different pulsed-field gel electrophoresis (PFGE) patterns and five E. coli isolates belonging to sequence type 117 (ST117) and phylogenetic group D were positive for fosA3 and all carried the blaCTX-M gene. In E. coli, the genetic structures IS26-ISEcp1-blaCTX-M-65-IS26-fosA3-1758 bp-IS26 and IS26-ISEcp1-blaCTX-M-3-blaTEM-1-IS26-fosA3-1758 bp-IS26 were present on transferable IncHI2/ST3 and F2:A-:B- plasmids, respectively. However, fosA3 was located on the chromosome of the seven P. mirabilis isolates. IS26-ISEcp1-blaCTX-M-65-IS26-fosA3-1758 bp-IS26 and IS26-blaCTX-M-14-611 bp-fosA3-1222 bp-IS26 were detected in three and four P. mirabilis isolates, respectively. Minicircles that contained both fosA3 and blaCTX-M-65 were shared between E. coli and P. mirabilis. This is the first report of the fosA3 gene integrated into the chromosome of P. mirabilis isolates with the blaCTX-M gene. The emergence and clonal spread of avian pathogenic E. coli ST117 with the feature of multidrug resistance and high virulence are a serious problem. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  11. Heat-stable Escherichia coli enterotoxin production in vivo.

    PubMed Central

    Whipp, S C; Moon, H W; Lyon, N C

    1975-01-01

    Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo. PMID:1097335

  12. The quantitative and condition-dependent Escherichia coli proteome

    PubMed Central

    Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

    2016-01-01

    Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

  13. YeeO from Escherichia coli exports flavins.

    PubMed

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

  14. YeeO from Escherichia coli exports flavins

    PubMed Central

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters. PMID:25482085

  15. Reassessing Escherichia coli as a cell factory for biofuel production.

    PubMed

    Wang, Chonglong; Pfleger, Brian F; Kim, Seon-Won

    2017-03-11

    Via metabolic engineering, industrial microorganisms have the potential to convert renewable substrates into a wide range of biofuels that can address energy security and environmental challenges associated with current fossil fuels. The user-friendly bacterium, Escherichia coli, remains one of the most frequently used hosts for demonstrating production of biofuel candidates including alcohol-, fatty acid- and terpenoid-based biofuels. In this review, we summarize the metabolic pathways for synthesis of these biofuels and assess enabling technologies that assist in regulating biofuel synthesis pathways and rapidly assembling novel E. coli strains. These advances maintain E. coli's position as a prominent host for developing cell factories for biofuel production.

  16. Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

    PubMed Central

    Toledo, M. Regina F.; Alvariza, M. do Carmo B.; Murahovschi, Jayme; Ramos, Sonia R. T. S.; Trabulsi, Luiz R.

    1983-01-01

    Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H−, O111ab:H2, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea. PMID:6339384

  17. Sources of Escherichia coli in a Coastal Subtropical Environment

    PubMed Central

    Solo-Gabriele, Helena M.; Wolfert, Melinda A.; Desmarais, Timothy R.; Palmer, Carol J.

    2000-01-01

    Sources of Escherichia coli in a coastal waterway located in Ft. Lauderdale, Fla., were evaluated. The study consisted of an extensive program of field measurements designed to capture spatial and temporal variations in E. coli concentrations as well as experiments conducted under laboratory-controlled conditions. E. coli from environmental samples was enumerated by using a defined substrate technology (Colilert-18). Field sampling tasks included sampling the length of the North Fork to identify the river reach contributing high E. coli levels, autosampler experiments at two locations, and spatially intense sampling efforts at hot spots. Laboratory experiments were designed to simulate tidal conditions within the riverbank soils. The results showed that E. coli entered the river in a large pulse during storm conditions. After the storm, E. coli levels returned to baseline levels and varied in a cyclical pattern which correlated with tidal cycles. The highest concentrations were observed during high tide, whereas the lowest were observed at low tide. This peculiar pattern of E. coli concentrations between storm events was caused by the growth of E. coli within riverbank soils which were subsequently washed in during high tide. Laboratory analysis of soil collected from the riverbanks showed increases of several orders of magnitude in soil E. coli concentrations. The ability of E. coli to multiply in the soil was found to be a function of soil moisture content, presumably due to the ability of E. coli to outcompete predators in relatively dry soil. The importance of soil moisture in regulating the multiplication of E. coli was found to be critical in tidally influenced areas due to periodic wetting and drying of soils in contact with water bodies. Given the potential for growth in such systems, E. coli concentrations can be artificially elevated above that expected from fecal impacts alone. Such results challenge the use of E. coli as a suitable indicator of water

  18. Structure of Water in Escherichia Coli B

    DTIC Science & Technology

    structure broadening of the NMR water spectrum. Using bacteria grown in the special chemically defined medium, we showed that the water in E. coli B was highly ordered and was very different from ’free’ water and from polywater .

  19. Slugs: Potential Novel Vectors of Escherichia coli O157

    PubMed Central

    Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

  20. 76 FR 58157 - Shiga Toxin-Producing Escherichia coli

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-20

    ... infections.\\1\\ \\1\\ U.S. Centers for Disease Control and Prevention. 2005. Shiga toxin-producing Escherichia coli (STEC). National Notifiable Diseases Surveillance System (NNDSS), 2005 Case Definition. http://www...) 1422-1429. \\6\\ Centers for Disease Control and Prevention. Bacterial Foodborne and Diarrheal Disease...

  1. Sequencing of Escherichia coli that cause persistent and transient Mastitis

    USDA-ARS?s Scientific Manuscript database

    The genomes of two strains of Escherichia coli that cause bovine mastitis were sequenced. These strains are known to be associated with persistent and transient mastitis: strain ECA-B causes a transient infection, and ECC-M leads to a persistent infection....

  2. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332

    PubMed Central

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E.; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos

    2017-01-01

    ABSTRACT   Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México. PMID:28232434

  3. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    EPA Science Inventory

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

  4. Escherichia coli as other Enterobacteriaceae: food poisoning and health effects

    USDA-ARS?s Scientific Manuscript database

    Many Escherichia coli strains are harmless, and they are an important commensal in the intestinal microflora; however, pathogenic strains also exist. The pathogenic strains can be divided into diarrhea-inducing strains and strains that reside in the intestines but only cause disease in bodily sites...

  5. Stringent control of FLP recombinase in Escherichia coli.

    PubMed

    Bowden, Steven D; Palani, Nagendra P; Libourel, Igor G L

    2017-02-01

    Site specific recombinases are invaluable tools in molecular biology, and are emerging as powerful recorders of cellular events in synthetic biology. We have developed a stringently controlled FLP recombinase system in Escherichia coli using an arabinose inducible promoter combined with a weak ribosome binding site.

  6. Enteroinvasive Escherichia coli severe dysentery complicated by rotavirus gastroenteritis.

    PubMed

    Pacheco-Gil, Leova; Ochoa, Theresa J; Flores-Romo, Leopoldo; DuPont, Herbert L; Estrada-Garcia, Teresa

    2006-11-01

    Enteroinvasive Escherichia coli (EIEC) is an important agent of pediatric diarrhea and dysentery in developing countries. We report a life-threatening severe dysentery case due to EIEC in a malnourished 4-month-old male, native Indian infant co-infected with rotavirus. The severe gastrointestinal bleeding anemia and hypovolemic shock was successfully treated with IV blood transfusions, rehydration and antibiotic therapy.

  7. Division Planes Alternate in Spherical Cells of Escherichia coli

    PubMed Central

    Begg, K. J.; Donachie, W. D.

    1998-01-01

    In the spherical cells of Escherichia coli rodA mutants, division is initiated at a single point, from which a furrow extends progressively around the cell. Using “giant” rodA ftsA cells, we confirmed that each new division furrow is initiated at the midpoint of the previous division plane and runs perpendicular to it. PMID:9573213

  8. More than a locomotive organelle: flagella in Escherichia coli.

    PubMed

    Zhou, Mingxu; Yang, Yang; Chen, Panlin; Hu, Huijie; Hardwidge, Philip R; Zhu, Guoqiang

    2015-11-01

    The flagellum is a locomotive organelle that allows bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions other than motility, including the relationship between flagella and bacterial virulence.

  9. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    EPA Science Inventory

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

  10. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    The increase in resistance rates to trimehtoprim-sulfamethoxazole (TMP/SMX) in isolates of Escherichia coli has become a matter of increasing concern. This has been particularly true in reference to community acquired urinary tract infections (UTI). This study utilized sewage i...

  11. Effect of phytoplankton on Escherichia coli survival in laboratory microcosms

    USDA-ARS?s Scientific Manuscript database

    Fecal contamination of water sources is an important water quality issue for agricultural irrigation ponds. Escherichia coli is a common microbial indicator used to evaluate recreational and irrigation water quality. Nuisance algae commonly grow in low- or no-flow irrigation water source The objecti...

  12. New types of Escherichia coli recombination-deficient mutants.

    PubMed

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  13. New types of Escherichia coli recombination-deficient mutants.

    PubMed Central

    Freifelder, D

    1976-01-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

  14. Escherichia coli growth studied by dual-parameter flow cytophotometry.

    PubMed Central

    Steen, H B; Boye, E

    1981-01-01

    The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s. PMID:7007339

  15. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    The increase in resistance rates to trimehtoprim-sulfamethoxazole (TMP/SMX) in isolates of Escherichia coli has become a matter of increasing concern. This has been particularly true in reference to community acquired urinary tract infections (UTI). This study utilized sewage i...

  16. Escherichia coli and other Enterobacteriaceae: Food poisoning and health effects

    USDA-ARS?s Scientific Manuscript database

    The family Enterobactericeae consists of rod-shaped, Gram-negative, facultatively anaerobic, non-spore forming bacteria and also includes the food-borne pathogens, Cronobacter spp., Escherichia coli, Salmonella enterica, Shigella spp., and Yersinia spp. Illness caused by these pathogens is acquired...

  17. Isolation of an Lc-specific Escherichia coli bacteriophage.

    PubMed Central

    Fralick, J A; Diedrich, D L; Casey-Wood, S

    1990-01-01

    We isolated an OmpF-specific bacteriophage whose host range mutant, SQ108h2, requires the presence of the Lc porin for its attachment and which can be used to screen or select for Lc-defective mutants among Escherichia coli K-12 strains lysogenic for the PA-2 converting phage. Images FIG. 1 PMID:1689719

  18. Plasmolysis of Escherichia coli B-r with sucrose.

    PubMed

    Scheie, P O

    1969-05-01

    Escherichia coli B/r cells were plasmolyzed in sucrose solutions and observed under phase contrast. The prevalence of plasmolysis under various conditions was noted, and the degree of plasmolysis was categorized as slight, extensive, or severe. The presence of ions reduced the prevalence of plasmolysis. Survival curves showed that extensive plasmolysis was not lethal to colony-forming ability.

  19. Naturally Occurring Extended-Spectrum Cephalosporinases in Escherichia coli

    PubMed Central

    Mammeri, Hedi; Poirel, Laurent; Fortineau, Nicolas; Nordmann, Patrice

    2006-01-01

    Genetic and functional characterization of the cephalosporinases produced by 65 clonally unrelated clinical Escherichia coli isolates revealed genetic diversity of the ampC genes and showed that Gln287, Cys287, Pro296, Leu298, and Phe350 substitutions were involved in extension of the hydrolysis spectrum to include ceftazidime and cefepime. PMID:16801449

  20. armA and aminoglycoside resistance in Escherichia coli.

    PubMed

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C; Moreno, Miguel A; Courvalin, Patrice; Domínguez, Lucas

    2005-06-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

  1. armA and Aminoglycoside Resistance in Escherichia coli

    PubMed Central

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C.; Courvalin, Patrice; Domínguez, Lucas

    2005-01-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant. PMID:15963296

  2. Norfloxacin resistance in a clinical isolate of Escherichia coli.

    PubMed Central

    Aoyama, H; Sato, K; Kato, T; Hirai, K; Mitsuhashi, S

    1987-01-01

    Analysis of DNA gyrase supercoiling and of norfloxacin uptake in Escherichia coli GN14176, a moderately norfloxacin-resistant clinical isolate, indicated that resistance was associated with both an altered drug target and a reduction in drug uptake. Images PMID:2829712

  3. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004.

    PubMed

    Qadri, Firdausi; Khan, Ashraful I; Faruque, Abu Syed G; Begum, Yasmin Ara; Chowdhury, Fahima; Nair, Gopinath B; Salam, Mohammed A; Sack, David A; Svennerholm, Ann-Mari

    2005-07-01

    Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.

  4. Plasmolysis of Escherichia coli B/r with Sucrose

    PubMed Central

    Scheie, Paul O.

    1969-01-01

    Escherichia coli B/r cells were plasmolyzed in sucrose solutions and observed under phase contrast. The prevalence of plasmolysis under various conditions was noted, and the degree of plasmolysis was categorized as slight, extensive, or severe. The presence of ions reduced the prevalence of plasmolysis. Survival curves showed that extensive plasmolysis was not lethal to colony-forming ability. PMID:4891252

  5. Multidrug-Resistant Escherichia coli in Bovine Animals, Europe

    PubMed Central

    Brennan, Evan; Martins, Marta; McCusker, Matthew P.; Wang, Juan; Alves, Bruno Martins; Hurley, Daniel; El Garch, Farid; Woehrlé, Frédérique; Miossec, Christine; McGrath, Leisha; Srikumar, Shabarinath; Wall, Patrick

    2016-01-01

    Of 150 Escherichia coli strains we cultured from specimens taken from cattle in Europe, 3 had elevated MICs against colistin. We assessed all 3 strains for the presence of the plasmid-mediated mcr-1 gene and identified 1 isolate as mcr-1–positive and co-resistant to β-lactam, florfenicol, and fluoroquinolone antimicrobial compounds. PMID:27533105

  6. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  7. Immunologic Control of Diarrheal Disease Due to Enterotoxigenic Escherichia coli

    DTIC Science & Technology

    1984-01-01

    Classical Enteropathogenic (Serotyped) Escherichia coli Strains of Proven Pathogenicity. Infect. Immun. 38:798-801, 1982. 8. Levine, M.M. Vacunas Contra...Microbiol., 18:808-815, 1983. 8 15. Levine, M.M., Lanata, C. Progresos en Vacunas Contra Diarrea Bacteriana. Adelantos Microbiol. Enferm. Inf., 2:67-117

  8. EcoCyc: Encyclopedia of Escherichia coli genes and metabolism.

    PubMed

    Karp, P D; Riley, M; Paley, S M; Pellegrini-Toole, A; Krummenacker, M

    1998-01-01

    The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli. The database describes 3030 genes of E.coli , 695 enzymes encoded by a subset of these genes, 595 metabolic reactions that occur in E.coli, and the organization of these reactions into 123 metabolic pathways. The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways. EcoCyc can be thought of as an electronic review article because of its copious references to the primary literature, and as a (qualitative) computational model of E.coli metabolism. EcoCyc is available at URL http://ecocyc.PangeaSystems.com/ecocyc/

  9. Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples

    PubMed Central

    Morcatti Coura, Fernanda; Diniz, Soraia de Araújo; Silva, Marcos Xavier; Mussi, Jamili Maria Suhet; Barbosa, Silvia Minharro; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2015-01-01

    This study analyzes the occurrence and distribution of phylogenetic groups of 391 strains of Escherichia coli isolated from poultry, cattle, and water buffalo. The frequency of the phylogroups was A = 19%, B1 = 57%, B2 = 2.3%, C = 4.6%, D = 2.8%, E = 11%, and F = 3.3%. Phylogroups A (P < 0.001) and F (P = 0.018) were associated with E. coli strains isolated from poultry, phylogroups B1 (P < 0.001) and E (P = 0.002) were associated with E. coli isolated from cattle, and phylogroups B2 (P = 0.003) and D (P = 0.017) were associated with E. coli isolated from water buffalo. This report demonstrated that some phylogroups are associated with the host analyzed and the results provide knowledge of the phylogenetic composition of E. coli from domestic animals. PMID:26421310

  10. Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium.

    PubMed

    Zhulin, I B; Rowsell, E H; Johnson, M S; Taylor, B L

    1997-05-01

    Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E. coli. The threshold of the attractant response in both species was 10(-6) M glycerol. Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential. Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished. Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E. coli or S. typhimurium but is involved in the negative chemotaxis of E. coli to high concentrations of glycerol. We propose that positive chemotaxis to glycerol in E. coli and S. typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level.

  11. Essential validation methods for E. coli strains created by chromosome engineering.

    PubMed

    Tiruvadi Krishnan, Sriram; Moolman, M Charl; van Laar, Theo; Meyer, Anne S; Dekker, Nynke H

    2015-01-01

    Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome. Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check. The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non

  12. Using zebra mussels to monitor Escherichia coli in environmental waters.

    PubMed

    Selegean, J P; Kusserow, R; Patel, R; Heidtke, T M; Ram, J L

    2001-01-01

    Use of the zebra mussel (Dreissena polymorpha) as an indicator of previously elevated bacteria concentrations in a watershed was examined. The ability of the zebra mussel to accumulate and purge Escherichia coli over several days was investigated in both laboratory and field experiments. In laboratory experiments, periodic enumeration of E. coli in mussels that had been exposed to a dilute solution of raw sewage demonstrated that (i) maximum concentrations of E. coli are reached within a few hours of exposure to sewage, (ii) the tissue concentration attained is higher than the concentration in the ambient water, and (iii) the E. coli concentrations take several days to return to preexposure concentrations when mussels are subsequently placed in sterile water. In field experiments conducted in southeast Michigan in the Clinton River watershed, brief increases in E. coli concentrations in the water were accompanied by increases in mussel concentrations of E. coli that lasted 2 or 3 d. The ability of mussels to retain and to concentrate E. coli made it possible to detect E. coli in the environment under conditions that conventional monitoring may often miss. Sampling caged mussels in a river and its tributaries may enable watershed managers to reduce the sampling frequency normally required to identify critical E. coli sources, thereby providing a more cost-effective river monitoring strategy for bacterial contamination.

  13. Cytotoxic Escherichia coli strains encoding colibactin colonize laboratory mice.

    PubMed

    García, Alexis; Mannion, Anthony; Feng, Yan; Madden, Carolyn M; Bakthavatchalu, Vasudevan; Shen, Zeli; Ge, Zhongming; Fox, James G

    2016-12-01

    Escherichia coli strains have not been fully characterized in laboratory mice and are not currently excluded from mouse colonies. Colibactin (Clb), a cytotoxin, has been associated with inflammation and cancer in humans and animals. We performed bacterial cultures utilizing rectal swab, fecal, and extra intestinal samples from clinically unaffected or affected laboratory mice. Fifty-one E. coli were isolated from 45 laboratory mice, identified biochemically, and selected isolates were serotyped. The 16S rRNA gene was amplified and sequenced for specific isolates, PCR used for clbA and clbQ gene amplification, and phylogenetic group identification was performed on all 51 E. coli strains. Clb genes were sequenced and selected E. coli isolates were characterized using a HeLa cell cytotoxicity assay. Forty-five of the 51 E. coli isolates (88%) encoded clbA and clbQ and belonged to phylogenetic group B2. Mouse E. coli serotypes included: O2:H6, O-:H-, OM:H+, and O22:H-. Clb-encoding O2: H6 mouse E. coli isolates were cytotoxic in vitro. A Clb-encoding E. coli was isolated from a clinically affected genetically modified mouse with cystic endometrial hyperplasia. Our findings suggest that Clb-encoding E. coli colonize laboratory mice and may induce clinical and subclinical diseases that may impact experimental mouse models.

  14. EFFECT OF DIHYDROSTREPTOMYCIN ON TETRAZOLIUM DYE REDUCTION IN ESCHERICHIA COLI

    PubMed Central

    Bragg, P. D.; Polglase, W. J.

    1963-01-01

    Bragg, P. D. (University of British Columbia, Vancouver, British Columbia, Canada) and W. J. Polglase. Effect of dihydrostreptomycin on tetrazolium dye reduction in Escherichia coli. J. Bacteriol. 85:795–800. 1963.—Sonic-disrupted extracts of Escherichia coli, grown without added antibiotic (sensitive and resistant), contained (in supernatant of fraction centrifuged at 100,000 × g) a dihydrostreptomycin-inhibitable, succinate-triphenyltetrazolium chloride (TTC) reductase activity. The succinate-TTC reductase activities of extracts of E. coli grown in the presence of dihydrostreptomycin (resistant and dependent) were relatively low and were not inhibited by the antibiotic. At a moderate magnesium concentration, the degree of inhibition by dihydrostreptomycin of succinate-TTC reductase activity was sufficiently marked to indicate an important site of action of the antibiotic. Magnesium, putrescine, and spermidine antagonized the action of dihydrostreptomycin in the succinate-TTC reductase system. PMID:14044945

  15. EFFECT OF DIHYDROSTREPTOMYCIN ON TETRAZOLIUM DYE REDUCTION IN ESCHERICHIA COLI.

    PubMed

    BRAGG, P D; POLGLASE, W J

    1963-04-01

    Bragg, P. D. (University of British Columbia, Vancouver, British Columbia, Canada) and W. J. Polglase. Effect of dihydrostreptomycin on tetrazolium dye reduction in Escherichia coli. J. Bacteriol. 85:795-800. 1963.-Sonic-disrupted extracts of Escherichia coli, grown without added antibiotic (sensitive and resistant), contained (in supernatant of fraction centrifuged at 100,000 x g) a dihydrostreptomycin-inhibitable, succinate-triphenyltetrazolium chloride (TTC) reductase activity. The succinate-TTC reductase activities of extracts of E. coli grown in the presence of dihydrostreptomycin (resistant and dependent) were relatively low and were not inhibited by the antibiotic. At a moderate magnesium concentration, the degree of inhibition by dihydrostreptomycin of succinate-TTC reductase activity was sufficiently marked to indicate an important site of action of the antibiotic. Magnesium, putrescine, and spermidine antagonized the action of dihydrostreptomycin in the succinate-TTC reductase system.

  16. Polyerositis and Arthritis Due to Escherichia coli in Gnotobiotic Pigs

    PubMed Central

    Waxler, G. L.; Britt, A. L.

    1972-01-01

    Forty gnotobiotic pigs from six litters were exposed orally to Escherichia coli 083:K·:NM at 69 to 148 hours of age, while 17 pigs from the same litters served as unexposed controls. Clinical signs of infection included fever, anorexia, diarrhea, lameness, and reluctance to move. Eighty-four percent of the exposed pigs in four litters died, while only 13% in two litters died. Gross and microscopic lesions included serofibrinous to fibrinopurulent polyserositis in 96% of the exposed pigs in four litters and 33% of the exposed pigs in two litters. A few pigs had gross and/or microscopic lesions of arthritis. Escherichia coli was routinely isolated from the serous and synovial cavities of infected pigs. Anti-hog cholera serum administered orally as a colostrum substitute gave partial protection against E. coli infection. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:4261837

  17. Mutations in Escherichia coli that effect sensitivity to oxygen

    SciTech Connect

    Jamison, C.S.; Adler, H.I.

    1987-11-01

    Fifteen oxygen-sensitive (Oxy/sup s/) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxy/sup s/-6, Oxy/sup s/-14, and Oxy/sup s/-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggest that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent.

  18. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

  19. Genetic engineering of probiotic Escherichia coli Nissle 1917 for clinical application.

    PubMed

    Ou, Bingming; Yang, Ying; Tham, Wai Liang; Chen, Lin; Guo, Jitao; Zhu, Guoqiang

    2016-10-01

    Escherichia coli strain Nissle 1917 (EcN) has been used as a probiotic. Genetic engineering has enhanced the utility of EcN in several vaccine and pharmaceutical preparations. We discuss in this mini review the genetics and physical properties of EcN. We also discuss the numerous genetic engineering strategies employed for EcN-based vaccine development, including recombinant plasmid transfer, genetic engineering of cryptic plasmids or the EcN chromosome, EcN bacterial ghosts and its outer membrane vesicles. We also provide a current update on the progress and the challenges regarding the use of EcN in vaccine development.

  20. Adhesive threads of extraintestinal pathogenic Escherichia coli.

    PubMed

    Antão, Esther-Maria; Wieler, Lothar H; Ewers, Christa

    2009-12-10

    The ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Long hair like extracellular appendages called fimbriae, produced by most Gram-negative pathogens, mediate specific attachment to the epithelial cell surface. Associated with the fimbriae is a protein called an adhesin, which directs high-affinity binding to specific cell surface components. In the last couple of years, an enormous amount of research has been undertaken that deals with understanding how bacterial pathogens adhere to host cells. E. coli in all probability is one of the best studied free-living organisms. A group of E. coli called Extraintestinal pathogenic E. coli (ExPEC) including both human and animal pathogens like Uropathogenic E. coli (UPEC), Newborn meningitic E. coli (NMEC) and Avian pathogenic E. coli (APEC), have been found to harbour many fimbriae including Type 1 fimbriae, P fimbriae, curli fibres, S fimbriae, F1C fimbriae, Dr fimbriae, afimbrial adhesins, temperature-sensitive haemagglutinin and many novel adhesin gene clusters that have not yet been characterized. Each of these adhesins is unique due to the recognition of an adhesin-specific receptor, though as a group these adhesins share common genomic organization. A newly identified putative adhesin temporarily termed ExPEC Adhesin I, encoded by gene yqi, has been recently found to play a significant role in the pathogenesis of APEC infection, thus making it an interesting candidate for future research. The aim of this review is to describe the role of ExPEC adhesins during extraintestinal infections known till date, and to suggest the idea of investigating their potential role in the colonization of the host gut which is said to be a reservoir for ExPEC.

  1. Experimental Escherichia coli O157:H7 carriage in calves.

    PubMed Central

    Brown, C A; Harmon, B G; Zhao, T; Doyle, M P

    1997-01-01

    Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding. PMID:8979335

  2. Travelers' diarrhea and toxigenic Escherichia coli.

    PubMed

    Gorbach, S L; Kean, B H; Evans, D G; Evans, D J; Bessudo, D

    1975-05-01

    In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.

  3. Genomic Comparative Study of Bovine Mastitis Escherichia coli

    PubMed Central

    Kempf, Florent; Slugocki, Cindy; Blum, Shlomo E.; Leitner, Gabriel; Germon, Pierre

    2016-01-01

    Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes. PMID:26809117

  4. Abundance of culturable versus viable Escherichia coli in freshwater.

    PubMed

    Servais, Pierre; Prats, Josué; Passerat, Julien; Garcia-Armisen, Tamara

    2009-07-01

    Approved methods traditionally used for Escherichia coli enumeration in waters are culture-based. However, these methods can underestimate the E. coli abundance in aquatic systems because they do not take into account cells that remain viable but have lost the ability to grow in or on culture media. We investigated, in freshwater samples, the abundance of (i) culturable E. coli, enumerated by the most probable number microplate method and (ii) viable E. coli, estimated using a procedure called DVC-FISH, which couples fluorescent in situ hybridization (FISH) and a viability testing technique (direct viable count (DVC)). The ratio of culturable to viable E. coli was close to 1 in highly contaminated waters (samples with a high concentration of culturable E. coli), but decreased drastically for weakly contaminated samples. This indicates a large fraction of viable but nonculturable (VBNC) E. coli in the latter samples. Microcosm experiments showed that some environmental factors, such as nutrient scarcity and solar irradiation, could lead to the presence of a high proportion of VBNC E. coli.

  5. Estimation of Escherichia coli in raw ground beef.

    PubMed Central

    Stiles, M E; Ng, L K

    1980-01-01

    This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference. PMID:7008695

  6. Genomic Comparative Study of Bovine Mastitis Escherichia coli.

    PubMed

    Kempf, Florent; Slugocki, Cindy; Blum, Shlomo E; Leitner, Gabriel; Germon, Pierre

    2016-01-01

    Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.

  7. [Acute diarrheal disease caused by enteropathogenic Escherichia coli in Colombia].

    PubMed

    Gómez-Duarte, Oscar G

    2014-10-01

    Intestinal Escherichia coli pathogens are leading causes of acute diarrheal disease in children less than 5 years in Latin America, Africa and Asia and a leading cause of death in children living in poorest communities in Africa and South East Asia. Studies on the role of E. coli pathogens in childhood diarrhea in Colombia and other countries in Latin America are limited due to the lack of detection assays in clinical laboratories at the main urban medical centers. Recent studies report that enterotoxigenic E. coli is the most common E. coli pathogens associated with diarrhea in children less than 5 years of age. Other E. coli pathotypes have been detected in children with diarrhea including enteropathogenic, enteroaggregative, shiga-toxin producing and diffusely adherent E. coli. It was also found that meat and vegetables at retail stores are contaminated with Shiga-toxin producing E. coli and enteroaggregative E. coli, suggesting that food products are involved in transmission and infection of the susceptible host. More studies are necessary to evaluate the mechanisms of transmission, the impact on the epidemiology of diarrheal disease, and management strategies and prevention of these pathogens affecting the pediatric population in Colombia.

  8. Introduction of quinolone resistant Escherichia coli to Swedish broiler population by imported breeding animals.

    PubMed

    Börjesson, Stefan; Guillard, Thomas; Landén, Annica; Bengtsson, Björn; Nilsson, Oskar

    2016-10-15

    During recent years a rapid increase of quinolone resistant Escherichia coli have been noted in the Swedish broiler population, despite the lack of a known selective pressure. The current study wanted to investigate if imported breeding birds could be a source for the quinolone resistant E. coli. The occurrence of quinolone resistant E. coli was investigated, using selective cultivation with nalidixic acid, in grand-parent birds on arrival to Sweden and their progeny. In addition, sampling in hatcheries and empty cleaned poultry houses was performed. Clonality of isolates was investigated using a 10-loci multiple-locus variable number tandem repeat analysis (MLVA). To identify the genetic basis for the resistance isolates were also analysed for occurrence of plasmid-mediated quinolone resistance (PMQR) determinants and characterization of chromosomal mutations. E. coli resistant to nalidixic acid occurred in grandparent birds imported to Sweden for breeding purposes. Four predominant MLVA types were identified in isolates from grandparent birds, parent birds and broilers. However, resistant E. coli with identical MLVA patterns were also present in hatcheries and poultry houses suggesting that the environment plays a role in the occurrence. Nalidixic acid resistance was due to a mutation in the gyrA gene and no PMQR could be identified. The occurrence of identical clones in all levels of the production pyramid points to that quinolone resistant E. coli can be introduced through imported breeding birds and spread by vertical transmission to all levels of the broiler production pyramid.

  9. Interaction of Type IV Toxin/Antitoxin Systems in Cryptic Prophages of Escherichia coli K-12

    PubMed Central

    Wen, Zhongling; Wang, Pengxia; Sun, Chenglong; Guo, Yunxue; Wang, Xiaoxue

    2017-01-01

    Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of “lemon-shaped” cells. Prolonged overproduction of YkfI led to the formation of “gourd-shaped” cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli, but ectopic expression in Pseudomonas and Shewanella did not cause the formation of “lemon-shaped” cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli. PMID:28257056

  10. Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections

    PubMed Central

    CHANG, Shao-Kuang; LO, Dan-Yuan; WEI, Hen-Wei; KUO, Hung-Chih

    2014-01-01

    This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried blaTEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs. PMID:25720807

  11. A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407▿ †

    PubMed Central

    Crossman, Lisa C.; Chaudhuri, Roy R.; Beatson, Scott A.; Wells, Timothy J.; Desvaux, Mickael; Cunningham, Adam F.; Petty, Nicola K.; Mahon, Vivienne; Brinkley, Carl; Hobman, Jon L.; Savarino, Stephen J.; Turner, Susan M.; Pallen, Mark J.; Penn, Charles W.; Parkhill, Julian; Turner, A. Keith; Johnson, Timothy J.; Thomson, Nicholas R.; Smith, Stephen G. J.; Henderson, Ian R.

    2010-01-01

    In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published. PMID:20802035

  12. Yeast DNA sequences initiating gene expression in Escherichia coli.

    PubMed

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  13. Proton-linked D-xylose transport in Escherichia coli.

    PubMed Central

    Lam, V M; Daruwalla, K R; Henderson, P J; Jones-Mortimer, M C

    1980-01-01

    The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli. PMID:6995439

  14. Escherichia coli control in a surface flow treatment wetland.

    PubMed

    MacIntyre, M E; Warner, B G; Slawson, R M

    2006-06-01

    A field experiment showed that numbers of Escherichia coli declined significantly when floating Lemna spp. plants were removed to create open water areas in a typical newly constructed surface flow treatment wetland in southern Ontario. It is suggested that E. coli declined immediately after Lemna removal because the Lemna was shading the water column from penetration by natural UV radiation, it was providing favourable attachment sites for the E. coli, and it was not allowing effective free exchange of oxygen from surface winds to the water column to maintain high enough dissolved oxygen supplies for predator zooplankton populations. Operators of wetland systems must have the specialized skills required to recognize the cause and the appropriate maintenance requirements to maintain efficient operation of such unconventional systems should E. coli numbers increase during the course of operation.

  15. Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli.

    PubMed

    Lee, Hyejin; Kim, Bong Gyu; Kim, Mihyang; Ahn, Joong-Hoon

    2015-09-01

    The flavonoid apigenin and its O-methyl derivative, genkwanin, have various biological activities and can be sourced from some vegetables and fruits. Microorganisms are an alternative for the synthesis of flavonoids. Here, to synthesize genkwanin from tyrosine, we first synthesized apigenin from p-coumaric acid using four genes (4CL, CHS, CHI, and FNS) in Escherichia coli. After optimization of different combinations of constructs, the yield of apigenin was increased from 13 mg/l to 30 mg/l. By introducing two additional genes (TAL and POMT7) into an apigenin-producing E. coli strain, we were able to synthesize 7-O-methyl apigenin (genkwanin) from tyrosine. In addition, the tyrosine content in E. coli was modulated by overexpressing aroG and tyrA. The engineered E. coli strain synthesized approximately 41 mg/l genkwanin.

  16. Escherichia coli early-onset sepsis: trends over two decades.

    PubMed

    Mendoza-Palomar, Natalia; Balasch-Carulla, Milena; González-Di Lauro, Sabina; Céspedes, Maria Concepció; Andreu, Antònia; Frick, Marie Antoinette; Linde, Maria Ángeles; Soler-Palacin, Pere

    2017-08-02

    Escherichia coli early-onset sepsis (EOS) is an important cause of mortality and morbidity in neonates, especially in preterm and very low birth weight (VLBW) newborns. The aim of our study was to evaluate potential changes in the clinical and microbiological characteristics of E. coli EOS in our setting. Epidemiological, clinical, and microbiological data from all neonates with proven E. coli EOS from January 1994 to December 2014 were retrospectively collected in a single tertiary care hospital in Barcelona (Spain). Seventy-eight E. coli EOS cases were analyzed. A slight increase in the incidence of E. coli EOS was observed during the study period. VLBW newborns remained the group with higher incidence (10.4 cases per 1000 live births) and mortality (35.3%). Systematic use of PCR increased E. coli EOS diagnosis, mainly in the term newborn group. There was an increase in resistant E. coli strains causing EOS, with especially high resistance to ampicillin and gentamicin (92.8 and 28.6%, respectively). Nonetheless, resistant strains were not associated with poorer clinical outcomes. There is an urgent need to reconsider the empirical therapy used in neonatal EOS, particularly in VLBW newborns. What is Known: • E. coli early-onset sepsis (EOS) and E. coli resistant strains have been described as overall stable but increasing in VLBW neonates (< 1.500 g) in previous studies. What is New: • Our study shows an increasing incidence of E. coli EOS in all age groups, overruling group B Streptoccocus for the last 10 years. E. coli resistant strains also increased equally in all age groups, with high resistance rates to our first line antibiotics (ampicillin and gentamicin). • Empiric antibiotic therapy of EOS, mainly in VLBW newborns, should be adapted to this new scenario.

  17. Conformational Fluctuations of Chromosomal DNA in E. coli

    NASA Astrophysics Data System (ADS)

    Freeman, Clarissa; Meiners, Jens-Christian

    2012-02-01

    We measured the conformational fluctuations of the bacterial chromosome in E. Coli in vivo using fluorescence correlation spectroscopy (FCS). The chromosomal DNA was randomly decorated with a cell-permeable intercalating dye. Conformational fluctuations of the DNA move the fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam. The time correlation function of the fluorescence intensity reflects the underlying dynamics of the DNA on length scales down to ˜200 nm. A comparison between live cells and dead yet structurally intact cells shows identical fluctuation spectra for short time scales, yet substantial differences for frequencies below 100 Hz. Live cells show much stronger fluctuations in this regime. This observation points to the crucial importance of active molecular motor action, as opposed to passive thermal noise, in driving larger conformational fluctuations in the chromosomal DNA, in particular on length scales exceeding ˜500 nm.

  18. [Production of L-lactic acid from pentose by a genetically engineered Escherichia coli].

    PubMed

    Zhao, Jinfang; Xu, Liyuan; Wang, Yongze; Zhao, Xiao; Wang, Jinhua

    2013-04-04

    In this study, we constructed a recombinant Escherichia coli strain for the production of high-purity L-lactic acid, using a homoethanol fermenting mutant E. coli SZ470 (deltafrdBC deltaldhA deltaackA deltafocA-pflB deltapdhR: :pflBp6-pflBrbs-aceEF-lpd) as the starting strain. By using homologous recombination, we deleted the adhE gene from SZ470 to obtain a mutant Escherichia coli JH01, which could not grow under anaerobic conditions. Then we cloned the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici and inserted it into the chromosome of JH01 via electroporation to obtain a recombinant strain Escherichia coli JH12. We evaluated the L-lactic acid production of the recombinant strain in a 15 L fermenter. In 10 L LB medium supplemented with 6% glucose, JH12 maintained maximal cell growth and an efficient L-lactic acid production rate for 36 h. Glucose consumption rate achieved was 1.46 g/(L x h) and L-lactic acid production rate was 1.14 g/(L x h). The results also show that 41.13 g/L lactic acid was produced, achieving a purity of 95.69% (based on total fermentation products). Xylose consumption rate was 0.88 g/(L x h) and L-lactic acid production rate was 0.60 g/(L x h). The production of lactic acid was 34.73 g/L, achieving a purity of 98%. There were no succinic acid and formic acid detected and only little amount of acetic acid generated during the fermentation. We constructed a homolactic acid fermentation strain E. coli JH12, which could efficiently convert glucose and xylose into high-purity L-lactic acid. JH12 could have great potential in industrial fermentation for L-lactic acid production.

  19. Resistance and virulence factors of Escherichia coli isolated from chicken.

    PubMed

    Pavlickova, Silvie; Dolezalova, Magda; Holko, Ivan

    2015-01-01

    Chicken meat has become an important part of the human diet and besides contamination by pathogenic Escherichia coli there is a risk of antibiotic resistance spreading via the food chain. The purpose of this study was to examine the prevalence of resistance against eight antibiotics and the presence of 14 virulence factors among 75 Escherichia coli strains isolated from chicken meat in the Czech Republic after classification into phylogenetic groups by the multiplex PCR method. More than half of strains belonged to A phylogroup, next frequently represented was B1 phylogroup, which suggests the commensal strains. The other strains were classified into phylogroups B2 and D, which had more virulence factors. Almost half of all E. coli strains were resistant to at least one of eight-tested antibiotics. A multidrug resistance was observed in 13% of strains. The most prevalent virulence genes were iucD, iss and tsh. None of genes encoding toxins was detected. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of analysis of virulence factors, antibiotic resistance and phylogroups assignment. It can provide a useful tool for prediction of a potential risk from food contaminated by E. coli.

  20. Escherichia coli as a model active colloid: A practical introduction.

    PubMed

    Schwarz-Linek, Jana; Arlt, Jochen; Jepson, Alys; Dawson, Angela; Vissers, Teun; Miroli, Dario; Pilizota, Teuta; Martinez, Vincent A; Poon, Wilson C K

    2016-01-01

    The flagellated bacterium Escherichia coli is increasingly used experimentally as a self-propelled swimmer. To obtain meaningful, quantitative results that are comparable between different laboratories, reproducible protocols are needed to control, 'tune' and monitor the swimming behaviour of these motile cells. We critically review the knowledge needed to do so, explain methods for characterising the colloidal and motile properties of E. coli cells, and propose a protocol for keeping them swimming at constant speed at finite bulk concentrations. In the process of establishing this protocol, we use motility as a high-throughput probe of aspects of cellular physiology via the coupling between swimming speed and the proton motive force.

  1. PROPERTIES OF A BACTERIOPHAGE DERIVED FROM ESCHERICHIA COLI K235

    PubMed Central

    Jesaitis, Margeris A.; Hutton, John J.

    1963-01-01

    A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants. PMID:14029160

  2. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html.

  3. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed Central

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html. PMID:8594596

  4. Functional role of bdm during flagella biogenesis in Escherichia coli.

    PubMed

    Kim, Ji-Sun; Kim, Yu Jin; Seo, Sojin; Seong, Maeng-Je; Lee, Kangseok

    2015-03-01

    The biofilm-dependent modulation gene (bdm) has recently been shown to play a role in osmotic-induced formation of biofilm in Escherichia coli. In this study, we demonstrated that deletion of bdm results in down-regulation of flagella biosynthesis genes and, consequently, a defect in E. coli motility. In addition, we employed atomic force microscopy to confirm the absence of flagella-like structures on the surface of bdm-null cells. These findings indicate that bdm plays a key role in regulatory pathway for the formation of flagella.

  5. Nitric oxide donor-mediated killing of bioluminescent Escherichia coli.

    PubMed Central

    Virta, M; Karp, M; Vuorinen, P

    1994-01-01

    The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity. PMID:7695261

  6. Bacterial self-defence: how Escherichia coli evades serum killing.

    PubMed

    Miajlovic, Helen; Smith, Stephen G

    2014-05-01

    The ability to survive the bactericidal action of serum is advantageous to extraintestinal pathogenic Escherichia coli that gain access to the bloodstream. Evasion of the innate defences present in serum, including complement and antimicrobial peptides, involves multiple factors. Serum resistance mechanisms utilized by E. coli include the production of protective extracellular polysaccharide capsules and expression of factors that inhibit or interfere with the complement cascade. Recent studies have also highlighted the importance of structural integrity of the cell envelope in serum survival. These survival strategies are outlined in this review with particular attention to novel findings and recent insights into well-established resistance mechanisms.

  7. Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption.

    PubMed

    Richter, Katrin; Gescher, Johannes

    2014-06-01

    Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

  8. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  9. Further studies of Escherichia coli in babies after normal delivery

    PubMed Central

    Bettelheim, K. A.; Teoh-Chan, Ching Haan; Chandler, Mary E.; O'Farrell, Sheila M.; Rahamin, Layla; Shaw, Elizabeth J.; Shooter, R. A.

    1974-01-01

    Previous work showed that on the basis of O serotyping alone of Escherichia coli, the majority of babies acquired the same O serotype as was found in the stools of their respective mothers. Further characterization of the E. coli by H serotyping, determination of their antibiotic resistance and ability to ferment six carbohydrates showed that in the majority of cases the previous results were confirmed. In a minority of cases this further testing showed that the strains were not identical. In some instances a number of strains isolated from the same pair showed different combinations of the markers used. PMID:4608224

  10. Metabolic engineering of Escherichia coli for 1-butanol production.

    PubMed

    Atsumi, Shota; Cann, Anthony F; Connor, Michael R; Shen, Claire R; Smith, Kevin M; Brynildsen, Mark P; Chou, Katherine J Y; Hanai, Taizo; Liao, James C

    2008-11-01

    Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.

  11. Inducible repair of oxidative DNA damage in Escherichia coli.

    PubMed

    Demple, B; Halbrook, J

    Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

  12. Sedimentation and gravitational instability of Escherichia coli Suspension

    NASA Astrophysics Data System (ADS)

    Douarche, Carine; Salin, Dominique; Collaboration between Laboratory FAST; LPS Collaboration

    2016-11-01

    The successive run and tumble of Escherichia coli bacteria provides an active matter suspension of rod-like particles with a large swimming diffusion. As opposed to inactive elongated particles, this diffusion prevents clustering and instability in the gravity field. We measure the time dependent E . coli concentration profile during their sedimentation. After some hours, due to the dioxygen consumption, a motile / non-motile front forms leading to a Rayleigh-Taylor type gravitational instability. Analyzing both sedimentation and instability in the framework of active particle suspensions, we can measure the relevant bacteria hydrodynamic characteristics such as its single particle sedimentation velocity and its hindrance volume.

  13. [Drug resistance of Escherichia coli strains isolated from poultry].

    PubMed

    Giurov, B; Korudzhiĭski, N; Bineva, I

    1981-01-01

    Studied was the sensitivity of a total of 143 strains of Escherichia coli, isolated from young birds and broilers died from coli septicaemia, to antibiotics and chemotherapeutics. The following descending order was established: gentamycin, carbenicillin, ampicillin, furazolidon, borgal, kanamycin, strep tomycin, chloramphenicol, neomycin sulphathiazole, and tetracycline. Markers of resistance were established with all strains with regard to the therapeutic agents in current and prospective use in industrial poultry farming. It is stated that a preliminary antibiogram is indispensable in order to obtain dependable results in the treatment of animals affected with colibacteriosis. An alternative is to apply directly those drugs to which the strains have shown highest sensitivity.

  14. SCO-1, a novel plasmid-mediated class A beta-lactamase with carbenicillinase characteristics from Escherichia coli.

    PubMed

    Papagiannitsis, C C; Loli, A; Tzouvelekis, L S; Tzelepi, E; Arlet, G; Miriagou, V

    2007-06-01

    A novel class A beta-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with beta-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8).

  15. Advances in molecular serotyping and subtyping of Escherichia coli

    DOE PAGES

    Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; ...

    2016-05-03

    Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

  16. Inactivation of Escherichia coli using atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Kuwahata, Hiroshi; Yamaguchi, Takeshi; Ohyama, Ryu-ichiro; Ito, Atsushi

    2015-01-01

    An atmospheric-pressure argon (Ar) plasma jet was applied to the inactivation of Escherichia coli. The Ar plasma jet was generated at a frequency of 10 kHz, an applied voltage of 10 kV, and an Ar gas flow rate of 10 L/min at atmospheric pressure. E. coli cells seeded on an agar medium in a Petri dish were inactivated by Ar plasma jet irradiation for 1 s. Scanning electron microscopy (SEM) revealed that E. coli cells were killed because their cell wall and membrane were disrupted. To determine the causes of the disruption of the cell wall and membrane of E. coli, we performed the following experiments: the measurement of the surface temperature of an agar medium using a thermograph, the analysis of an emission spectrum of a plasma jet obtained using a multichannel spectrometer, and the determination of the distribution of the concentration of hydrogen peroxide (H2O2) generated on an agar medium by plasma jet irradiation using semiquantitative test strips. Moreover, H2O2 solutions of different concentrations were dropped onto an agar medium seeded with E. coli cells to examine the contribution of H2O2 to the death of E. coli. The results of these experiments showed that the cell wall and membrane of E. coli were disrupted by electrons in the plasma jet, as well as by electroneutral excited nitrogen molecules (N2) and hydroxyl (OH) radicals in the periphery of the plasma jet.

  17. Gentamicin resistance among Escherichia coli strains isolated in neonatal sepsis.

    PubMed

    Hasvold, J; Bradford, L; Nelson, C; Harrison, C; Attar, M; Stillwell, T

    2013-01-01

    Neonatal sepsis is a significant cause of morbidity and mortality among term and preterm infants. Ampicillin and gentamicin are standard empiric therapy for early onset sepsis. Four cases of neonatal sepsis secondary to Escherichia coli (E. coli) found to be gentamicin resistant occurred within a five week period in one neonatal intensive care unit (NICU). To determine whether these cases could be tied to a single vector of transmission, and to more broadly evaluate the incidence of gentamicin resistant strains of E. coli in the neonatal population at our institution compared to other centers, we reviewed the charts of the four neonates (Infants A through D) and their mothers. The E. coli isolates were sent for Pulse Field Gel Electrophoresis (PFGE) to evaluate for genetic similarity between strains. We also reviewed all positive E. coli cultures from one NICU over a two year period. Infants A and B had genetically indistinguishable strains which matched that of urine and placental cultures of Infant B's mother. Infant C had a genetically distinct organism. Infant D, the identical twin of Infant C, did not have typing performed. Review of all cultures positive for E. coli at our institution showed a 12.9 percent incidence of gentamicin-resistance. A review of other studies showed that rates of resistance vary considerably by institution. We conclude that gentamicin-resistant E. coli is a relatively uncommon cause of neonatal sepsis, but should remain a consideration in patients who deteriorate despite initiation of empiric antibiotics.

  18. The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

    PubMed

    Kogoma, T; von Meyenburg, K

    1983-01-01

    The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.

  19. The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

    PubMed Central

    Kogoma, T; von Meyenburg, K

    1983-01-01

    The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism. Images Fig. 2. PMID:11894964

  20. No evidence for a bovine mastitis Escherichia coli pathotype.

    PubMed

    Leimbach, Andreas; Poehlein, Anja; Vollmers, John; Görlich, Dennis; Daniel, Rolf; Dobrindt, Ulrich

    2017-05-08

    Escherichia coli bovine mastitis is a disease of significant economic importance in the dairy industry. Molecular characterization of mastitis-associated E. coli (MAEC) did not result in the identification of common traits. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype has been proposed suggesting virulence traits that differentiate MAEC from commensal E. coli. The present study was designed to investigate the MPEC pathotype hypothesis by comparing the genomes of MAEC and commensal bovine E. coli. We sequenced the genomes of eight E. coli isolated from bovine mastitis cases and six fecal commensal isolates from udder-healthy cows. We analyzed the phylogenetic history of bovine E. coli genomes by supplementing this strain panel with eleven bovine-associated E. coli from public databases. The majority of the isolates originate from phylogroups A and B1, but neither MAEC nor commensal strains could be unambiguously distinguished by phylogenetic lineage. The gene content of both MAEC and commensal strains is highly diverse and dominated by their phylogenetic background. Although individual strains carry some typical E. coli virulence-associated genes, no traits important for pathogenicity could be specifically attributed to MAEC. Instead, both commensal strains and MAEC have very few gene families enriched in either pathotype. Only the aerobactin siderophore gene cluster was enriched in commensal E. coli within our strain panel. This is the first characterization of a phylogenetically diverse strain panel including several MAEC and commensal isolates. With our comparative genomics approach we could not confirm previous studies that argue for a positive selection of specific traits enabling MAEC to elicit bovine mastitis. Instead, MAEC are facultative and opportunistic pathogens recruited from the highly diverse bovine gastrointestinal microbiota. Virulence-associated genes implicated in mastitis are a by-product of commensalism with the primary function

  1. EFFECT OF VISIBLE RANGE ELECTROMAGNETIC RADIATIONS ON ESCHERICHIA COLI.

    PubMed

    Azeemi, Samina T Yousuf; Shaukat, Saleem Farooq; Azeemi, Khawaja Shamsuddin; Khan, Idrees; Mahmood, Khalid; Naz, Farah

    2017-01-01

    Escherichia coli is the agent responsible for a range of clinical diseases. With emerging antimicrobial resistance, other treatment options including solar/photo-therapy are becoming increasingly common. Visible Range Radiation Therapy/Colour Therapy is an emerging technique in the field of energy/vibrational medicine that uses visible spectrum of Electromagnetic Radiations to cure different diseases. In this study, our goal was to understand the effect of Visible Range Electromagnetic Radiations on E. coli (in vitro) and therefore find out the most appropriate visible range radiation for the treatment of diseases caused by E. coli. A total of 6 non-repetitive E. coli isolates were obtained from urine samples obtained from hospitalized patients with UTI. Single colony of E. coli was inoculated in 3 ml of Lysogeny Broth (LB) and 40 μl of this E. coli suspension was poured into each of the plastic tubes which were then irradiated with six different wavelengths in the visible region (Table. 1) after 18 hours with one acting as a control. The Optical Densities of these irradiated samples were then measured. Furthermore, scanning electron microscopy (TEFCAN ZEGA3) was carried out. The analysis of the microscopic and SEM images of irradiated E. coli samples with six different visible range radiations is representative of The fact that E. coli responded differently to every applied radiation in the visible region and the most profound inhibitory effects were that of 538nm Visible Range Radiation (Green) which proved to be bactericidal and 590nm Visible Range Radiation (yellow) which was bacteriostatic. The enhanced growth of E. coli with varying degrees was clearly observed in 610nm (orange), 644nm (red), 464nm (Purple) and 453nm (blue). It can be concluded that 538nm (Green) and 590nm (Yellow) can effectively be used for treating E. coli borne diseases.

  2. Formation of nonculturable Escherichia coli in drinking water.

    PubMed

    Bjergbaek, L A; Roslev, P

    2005-01-01

    To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. The existence of substrate-responsive but nonculturable cells should be considered

  3. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (L-glycerol 3-phosphate: CMP phosphatidyltransferase) catalyzing the synthesis of phosphatidyl glycerophosphate from CDP-diglyceride and L-glycerol 3-phosphate has been rendered soluble by treatment of the particulate, membrane-containing fraction of E. coli with Triton X-100 and has been partially purified. The enzyme, devoid of phosphatidyl glycerophosphatase activity, is specific for L-glycerol 3-phosphate and is completely dependent upon added Mg(++) or Mn(++) for activity. It has high affinity for CDP-diglyceride and can be used for the assay of this nucleotide. Other properties of the enzyme are also described.

  4. Specific mistranslation in hisT mutants of Escherichia coli.

    PubMed

    Parker, J

    1982-01-01

    Certain strains of Escherichia coli mistranslate at very high frequencies when starved for asparagine or histidine. This mistranslation is the result of misreading events on the ribosome. The introduction of a hisT mutation into such a strain decreases the frequency of mistranslation during histidine starvation but not during asparagine starvation. The most likely explanation is that the replacement of the pseudouridine residue in the anticodon loop of glutamine specific transfer ribonucleic acid by uridine in hisT mutants leads to an increase in fidelity of transfer ribonucleic acid function. The hisT gene in Escherichia coli has also been more accurately mapped, giving the gene order purF-hisT-aroC-fadL-dsdA.

  5. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

    PubMed

    Tajkarimi, Mehrdad; Harrison, Scott H; Hung, Albert M; Graves, Joseph L

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.

  6. LYSIS OF ESCHERICHIA COLI BY SULFHYDRYL-BINDING REAGENTS

    PubMed Central

    Schaechter, M.; Santomassino, Katherine A.

    1962-01-01

    Schaechter, M. (College of Medicine, University of Florida, Gainesville) and K. Santomassino. Lysis of Escherichia coli by sulfhydryl-binding reagents. J. Bacteriol. 84:318–325. 1962—Washed suspensions of gram-negative rods were lysed by low concentrations of some sulfhydryl-binding and oxidizing reagents, but not by reducing agents. Some kinetic aspects of this phenomenon were studied with p-chloromercuribenzoate and Escherichia coli B/r. Structures resulting from the action of this reagent consisted of impure cell walls. These could be purified by treatment with trypsin. Cell walls prepared mechanically and cell membranes obtained by lysing protoplasts were not overtly affected by this chemical. Images PMID:14497913

  7. Enterotoxigenic Escherichia coli infection in captive black-footed ferrets.

    PubMed

    Bradley, G A; Orr, K; Reggiardo, C; Glock, R D

    2001-07-01

    Enterotoxigenic Escherichia coli with genes for heat stabile toxins Sta and STb was isolated from the gastrointestinal tract and multiple visceral organs of three adult and three juvenile black-footed ferrets (Mustela nigripes) that died in a captive breeding colony between 24 May 1998 and 2 July 1998. Similar isolates were obtained from rectal swabs of one adult and one juvenile that were clinically ill. All were fed a diet composed of mink chow, raw rabbit meat, beef liver powder, blood meal and lard. Escherichia coli of the same toxin genotype was isolated from the mixed ration. Clinical signs included sudden death, dehydration, anorexia and diarrhea. Necropsy lesions included acute enteritis with large numbers of rod shaped bacteria microscopically visible on intestinal villi.

  8. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  9. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  10. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

    PubMed Central

    Tajkarimi, Mehrdad; Harrison, Scott H.; Hung, Albert M.; Graves, Joseph L.

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism. PMID:26914334

  11. Thiolases of Escherichia coli: purification and chain length specificities.

    PubMed Central

    Feigenbaum, J; Schulz, H

    1975-01-01

    The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated. A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation. The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis. The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates. In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A. PMID:236278

  12. Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation.

    PubMed

    Pla, J; Sánchez, M; Palacios, P; Vicente, M; Aldea, M

    1991-07-01

    An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.

  13. Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine.

    PubMed Central

    Sullivan, M A; Cannon, J F; Webb, F H; Bock, R M

    1985-01-01

    Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL. These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E. coli chromosome. Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes. The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations. The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALys, was impaired in asuE mutant strains, presumably at the 2-thiolation step. Images PMID:3881393

  14. Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli.

    PubMed

    Keller, Rogéria; Ordoñez, Juana G; de Oliveira, Rosana R; Trabulsi, Luiz R; Baldwin, Thomas J; Knutton, Stuart

    2002-05-01

    O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

  15. Fosfomycin resistance plasmids do not affect fosfomycin transport into Escherichia coli.

    PubMed Central

    León, J; García-Lobo, J M; Ortiz, J M

    1982-01-01

    Escherichia coli cells carrying fosfomycin resistance plasmids were able to take up fosfomycin from the medium to the same extent as plasmid-free bacteria. The antibiotic entered the plasmid-harboring cells by means of the glpT and uhp transport systems, as is the case with susceptible bacteria. Active fosfomycin could be detected in soluble extracts of cells which had previously been incubated in the presence of the antibiotic. Furthermore, fosfomycin resistance plasmids did not confer on E. coli cells resistance to the novel antibiotic FR-31564, which is incorporated by the same transport systems as fosfomycin. We conclude that, in contrast to chromosomal resistance mutants, altered transport does not play a role in the plasmid-encoded fosfomycin resistance mechanism. PMID:7044304

  16. A simple and effective method for construction of Escherichia coli strains proficient for genome engineering.

    PubMed

    Ryu, Young Shin; Biswas, Rajesh Kumar; Shin, Kwangsu; Parisutham, Vinuselvi; Kim, Suk Min; Lee, Sung Kuk

    2014-01-01

    Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

  17. Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity.

    PubMed

    Amado, Luciana; Kuzminov, Andrei

    2013-11-15

    Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.

  18. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol.

    SciTech Connect

    Donnelly, M. I.; Millard, C. S.; Clark, D. P.; Chen, M. J.; Rathke, J. W.; Southern Illinois Univ.

    1998-04-01

    Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E.coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.

  19. Shear alters motility of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Jalali, Maryam; Sheng, Jian

    2013-11-01

    Understanding of locomotion of microorganisms in shear flows drew a wide range of interests in microbial related topics such as biological process including pathogenic infection and biophysical interactions like biofilm formation on engineering surfaces. We employed microfluidics and digital holography microscopy to study motility of E. coli in shear flows. We controlled the shear flow in three different shear rates: 0.28 s-1, 2.8 s-1, and 28 s-1 in a straight channel with the depth of 200 μm. Magnified holograms, recorded at 15 fps with a CCD camera over more than 20 minutes, are analyzed to obtain 3D swimming trajectories and subsequently used to extract shear responses of E.coli. Thousands of 3-D bacterial trajectories are tracked. The change of bacteria swimming characteristics including swimming velocity, reorientation, and dispersion coefficient are computed directly for individual trajectory and ensemble averaged over thousands of realizations. The results show that shear suppresses the bacterial dispersions in bulk but promote dispersions near the surface contrary to those in quiescent flow condition. Ongoing analyses are focusing to quantify effect of shear rates on tumbling frequency and reorientation of cell body, and its implication in locating the hydrodynamic mechanisms for shear enhanced angular scattering. NIH, NSF, GoMRI.

  20. Positive regulation of the Escherichia coli glycine cleavage enzyme system.

    PubMed Central

    Wilson, R L; Steiert, P S; Stauffer, G V

    1993-01-01

    A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv. PMID:8423160

  1. Lipophilic chelator inhibition of electron transport in Escherichia coli.

    PubMed Central

    Crane, R T; Sun, I L; Crane, F L

    1975-01-01

    The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli. The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect. Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity. Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited. Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition. PMID:1092663

  2. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

    PubMed

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos; Cevallos, Miguel A; Xicohtencatl-Cortes, Juan

    2017-02-23

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México. Copyright © 2017 Saldaña-Ahuactzi et al.

  3. Spurious hydrogen sulfide production by Providencia and Escherichia coli species.

    PubMed Central

    Treleaven, B E; Diallo, A A; Renshaw, E C

    1980-01-01

    Hydrogen sulfide production was noted in two Escherichia coli strands and one Provaidenica alcalifaciens (Proteus inconstans A) strain isolated from clinical stool specimens durin the summer of 1979. An investigation into this phenomenon revealed the predence of Eubacterium lentum, an anaerobe, growing in synergism with the Enterobacteriaceae and producing H2s. The implications of this association are discssed with reference to clinical microbiology laboratory practice. PMID:7000823

  4. Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli.

    PubMed

    Kong, Haishen; Hong, Xiaoping; Li, Xuefen

    2015-08-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that causes acute and persistent diarrhea in children and adults. While the pathogenic mechanisms of EAEC intestinal colonization have been uncovered (including bacterial adhesion, enterotoxin and cytotoxin secretion, and stimulation of mucosal inflammation), those of severe extraintestinal infections remain largely unknown. The recent emergence of multidrug resistant EAEC represents an alarming public health threat and clinical challenge, and research on the molecular mechanisms of resistance is urgently needed.

  5. Automated recombinant protein expression screening in Escherichia coli.

    PubMed

    Busso, Didier; Stierlé, Matthieu; Thierry, Jean-Claude; Moras, Dino

    2008-01-01

    To fit the requirements of structural genomics programs, new as well as classical methods have been adapted to automation. This chapter describes the automated procedure developed within the Structural Biology and Genomics Platform, Strasbourg for performing recombinant protein expression screening in Escherichia coli. The procedure consists of parallel competent cells transformation, cell plating, and liquid culture inoculation, implemented for up to 96 samples at a time.

  6. Antibacterial efficacy of silver nanoparticles against Escherichia coli

    NASA Astrophysics Data System (ADS)

    Pattabi, Rani M.; Thilipan, G. Arun Kumar; Bhat, Vinayachandra; Sridhar, K. R.; Pattabi, Manjunatha

    2013-02-01

    Silver nanoparticles (AgNPs) synthesized by subjecting an aqueous solution of AgNO3 and polyvinyl alcohol to irradiation from an UV lamp has been studied for its antibacterial potential against Gram-negative bacteria (Escherichia coli). The diameter of the zone of inhibition is found to depend on both the irradiation time and the nanoparticle concentration. As the synthesis method adopted uses no toxic reagents, these particles may serve as promising candidates in the search for better antibacterial agents.

  7. Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli.

    PubMed

    Gallagher, D T; Eisenstein, E; Fisher, K E; Zondlo, J; Chinchilla, D; Yu, H D; Dill, J; Winborne, E; Ducote, K; Xiao, G; Gilliland, G L

    1998-05-01

    The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms. Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions. Diffraction data sets to 2.8 A resolution (native) and 2. 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.

  8. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    PubMed Central

    Ireland, J C; Klostermann, P; Rice, E W; Clark, R M

    1993-01-01

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfection capabilities of the reactor. In water devoid of significant amounts of inorganic-radical scavengers, rapid cell death was observed with both pure cultures and members of the indigenous flora in a natural water sample. PMID:8390819

  9. Effects of Acridine Orange on the Growth of Escherichia coli

    PubMed Central

    Southwick, Frederick S.; Carr, Howard S.; Carden, George A.; D'Alisa, Rose M.; Rosenkranz, Herbert S.

    1972-01-01

    Exposure of Escherichia coli to critical acridine orange (AO) concentrations did not result in loss of viability. However, the deoxyribonucleic acid (DNA) of cells exposed to such agents was rapidly degraded and repolymerized. On the other hand, a bacterium deficient in DNA repair (pol A1−, lacking DNA polymerase) was sensitive to the action of AO. The DNA of such cells was also degraded but it was not repaired. PMID:4553001

  10. Escherichia coli adherence to HEp-2 cells with prefixed cells.

    PubMed Central

    Zepeda-Lopez, H M; Gonzalez-Lugo, G M

    1995-01-01

    We describe a new method which uses cold absolute methanol-prefixed cells for adherence of enteropathogenic Escherichia coli to HEp-2 cells. We found that a method using bacteria grown in Penassay broth to 10(6) to 10(7) CFU/ml and incubated with prefixed cells for 3 h at 37 degrees C, showed 100% sensitivity and specificity against a method using live cells. PMID:7615770

  11. Electric field induced bacterial flocculation of Enteroaggregative Escherichia coli 042

    SciTech Connect

    Kumar, Aloke; Mortensen, Ninell P; Mukherjee, Partha P; Retterer, Scott T; Doktycz, Mitchel John

    2011-01-01

    A response of the aggregation dynamics of enteroaggregative Escherichia coli under low magnitude steady and oscillating electric fields is presented. The presence of uniform electric fields hampered microbial adhesion and biofilm formation on a transverse glass surface, but instead promoted the formation of flocs. Extremely heterogeneous distribution of live and dead cells was observed among the flocs. Moreover, floc formation was largely observed to be independent of the frequency of alternating electric fields.

  12. Role for the female in bacterial conjugation in Escherichia coli.

    PubMed

    Freifelder, D

    1967-08-01

    Hfr and F' Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F' Lac or of lambda prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female.

  13. Role for the Female in Bacterial Conjugation in Escherichia coli

    PubMed Central

    Freifelder, David

    1967-01-01

    Hfr and F′ Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F′ Lac or of λ prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female. PMID:5341864

  14. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  15. Some factors affecting cyclopropane acid formation in Escherichia coli

    PubMed Central

    Knivett, V. A.; Cullen, Julia

    1965-01-01

    1. The fatty acid composition of the extractable lipids of Escherichia coli varied with growth conditions. 2. The principal fatty acids were palmitic acid, hexadecenoic acid, octadecenoic acid and the cyclopropane acids, methylenehexadecanoic acid and methyleneoctadecanoic acid. 3. Cyclopropane acid formation from monoenoic acids was increased by acid media, poor oxygen supply, or high growth temperature. 4. Cyclopropane acid formation was decreased by alkaline media, well oxygenated conditions, the presence of citrate, or lack of Mg2+. PMID:5324304

  16. Evidence of Pathogenic Subgroups among Atypical Enteropathogenic Escherichia coli Strains▿

    PubMed Central

    Scaletsky, Isabel C. A.; Aranda, Katia R. S.; Souza, Tamara B.; Silva, Neusa P.; Morais, Mauro B.

    2009-01-01

    We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea. PMID:19759223

  17. Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

    PubMed

    Watanabe, Jun; Tanaka, Hisaki; Akagawa, Takumi; Mogi, Yoshinobu; Yamazaki, Tatsuo

    2007-10-01

    To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.

  18. Two Forms of d-Glycerate Kinase in Escherichia coli

    PubMed Central

    Ornston, M. K.; Ornston, L. N.

    1969-01-01

    Escherichia coli K-12 synthesizes two chromatographically distinct forms of glycerate kinase which differ both in their thermolability and in the dependence of their activity upon pH. One enzymatic form, GK I, is found in cells grown with glycerate, glucarate, or glycolate. Of these compounds, glycolate is the only carbon source that elicits the synthesis of the second enzymatic form, GK II. PMID:4887503

  19. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation.

    PubMed

    Laverty, Garry; Gorman, Sean P; Gilmore, Brendan F

    2014-07-18

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

  20. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    PubMed Central

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014