Note: This page contains sample records for the topic bacterial rrna extraction from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: November 12, 2013.
1

Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning  

PubMed Central

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

Dunbar, John; Takala, Shannon; Barns, Susan M.; Davis, Jody A.; Kuske, Cheryl R.

1999-01-01

2

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

PubMed Central

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

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

2012-01-01

3

Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses  

Microsoft Academic Search

Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% a-Proteobacteria, 31% ß-Proteobacteria, 33% ?-Proteobacteria, and 2% d-Proteobacteria), with

Yanhe Ma; Weizhou Zhang; Yanfen Xue; Peijin Zhou; Antonio Ventosa; William D. Grant

2004-01-01

4

Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis  

PubMed Central

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

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

1999-01-01

5

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays  

SciTech Connect

ABSTRACT-We report on the development and validation of simple microarray method for the direct detection of intact 16S rRNA from un-purified soil extracts. Total RNA from Geobacter chapellei and Desulfovibrio desulfuricans was hybridized to an oligonculeotide array consisting of universal and species-specific 16S rRNA probes...

Small, Jack A.; Call, Douglas R.; Brockman, Fred J.; Straub, Tim M.; Chandler, Darrell P.

2001-10-01

6

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays  

PubMed Central

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 ?g of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

2001-01-01

7

Comparison of bacterial communities in the Solimões and Negro River tributaries of the Amazon River based on small subunit rRNA gene sequences.  

PubMed

The microbiota of the Amazon River basin has been little studied. We compared the structure of bacterial communities of the Solimões and Negro Rivers, the main Amazon River tributaries, based on analysis of 16S rRNA gene sequences. Water was sampled with a 3-L Van Dorn collection bottle; samples were collected at nine different points/depths totaling 27 L of water from each river. Total DNA was extracted from biomass retained by a 0.22-?m filter after sequential filtration of the water through 0.8- and 0.22-?m filters. The 16S rRNA gene was amplified by PCR, cloned and sequenced, and the sequences were analyzed with the PHYLIP and DOTUR programs to obtain the operational taxonomic units (OTUs) and to calculate the diversity and richness indices using the SPADE program. Taxonomic affiliation was determined using the naive Bayesian rRNA Classifier of the RDP II (Ribosomal Database Project). We recovered 158 sequences from the Solimões River grouped into 103 OTUs, and 197 sequences from the Negro River library grouped into 90 OTUs by the DOTUR program. The Solimões River was found to have a greater diversity of bacterial genera, and greater estimated richness of 446 OTUs, compared with 242 OTUs from the Negro River, as calculated by ACE estimator. The Negro River has less bacterial diversity, but more 16S rRNA gene sequences belonging to the bacterial genus Polynucleobacter were detected; 56 sequences from this genus were found (about 30% of the total sequences). We suggest that a more in-depth investigation be made to elucidate the role played by these bacteria in the river environment. These differences in bacterial diversity between Solimões and Negro Rivers could be explained by differences in organic matter content and pH of the rivers. PMID:22183948

Peixoto, J C C; Leomil, L; Souza, J V; Peixoto, F B S; Astolfi-Filho, S

2011-12-08

8

Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment?  

PubMed Central

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3? tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5? tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055T. Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.

Lu, Ting; Stroot, Peter G.; Oerther, Daniel B.

2009-01-01

9

Bacterial community analysis of a gas-phase biotrickling filter for biogas mimics desulfurization through the rRNA approach  

Microsoft Academic Search

The bacterial composition of a lab-scale biotrickling filter (BTF) treating high loads of H2S was investigated by the rRNA approach. Two 16S rRNA gene clone libraries were established 42 and 189 d after reactor startup, while fluorescent in-situ hybridization (FISH) with DNA probes was performed throughout 260d of reactor operation. Diversity, community structure and metamorphosis were studied from reactor startup

Juan P. Maestre; R. Rovira; F. J. Álvarez-Hornos; M. Fortuny; J. Lafuente; X. Gamisans; D. Gabriel

2010-01-01

10

Rapid diagnosis of sepsis and bacterial meningitis in children with real-time fluorescent quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene.  

PubMed

A method for the detection of bacterial pathogens in sepsis and bacterial meningitis with 16S rRNA gene- based real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) is developed. A total of 190 blood specimens and 5 cerebrospinal fluid specimens from neonates with suspected sepsis or bacterial meningitis were evaluated with 16S rRNA gene-based real-time FQ-PCR assay. The positive rate of the real-time FQ-PCR assay was significantly higher (25/195, 12.82%) than that of bacterial culture (15/195, 7.69%; P = .002). When bacterial culture was used as a control, the sensitivity of the real-time FQ-PCR was 100%, the specificity was 94.4%, and Youden's index was 0.944. This study suggests that 16S rRNA gene-based real-time FQ-PCR assay is an important and accurate method in the detection of bacterial pathogens of sepsis and bacterial meningitis and should have a promising usage in the diagnosis of sepsis and bacterial meningitis. PMID:19407210

Chen, Li-Hua; Duan, Qun-Jun; Cai, Mei-Ting; Wu, Yi-Dong; Shang, Shi-Qiang

2009-04-30

11

Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning  

Microsoft Academic Search

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated

JOHN DUNBAR; SHANNON TAKALA; SUSAN M. BARNS; JODY A. DAVIS; CHERYL R. KUSKE

1999-01-01

12

Systematic 16S rRNA Gene Sequencing of Atypical Clinical Isolates Identified 27 New Bacterial Species Associated with Humans  

PubMed Central

Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a ?1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (?10 cases reported in the literature) human pathogens. Eleven new species, “Actinobaculum massiliae,” “Candidatus Actinobaculum timonae,” Paenibacillus sanguinis, “Candidatus Bacteroides massiliae,” Chryseobacterium massiliae, “Candidatus Chryseobacterium timonae,” Paenibacillus massiliensis, “Candidatus Peptostreptococcus massiliae,” “Candidatus Prevotella massiliensis,” Rhodobacter massiliensis, and “Candidatus Veillonella atypica” were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases.

Drancourt, M.; Berger, P.; Raoult, D.

2004-01-01

13

Distinct Ectomycorrhizospheres Share Similar Bacterial Communities as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes  

PubMed Central

Analysis of the 16S rRNA gene sequences generated from Xerocomus pruinatus and Scleroderma citrinum ectomycorrhizospheres revealed that similar bacterial communities inhabited the two ectomycorrhizospheres in terms of phyla and genera, with an enrichment of the Burkholderia genus. Compared to the bulk soil habitat, ectomycorrhizospheres hosted significantly more Alpha-, Beta-, and Gammaproteobacteria.

Oger, P.; Morin, E.; Frey-Klett, P.

2012-01-01

14

PCR-DGGE detection of the bacterial community structure in the Inner Mongolia steppe with two different DNA extraction methods  

Microsoft Academic Search

Compared with conventional methods, molecular biological technique, such as PCR-DGGE (denaturing gradient gel electrophoresis), is informative in examining the structure of the soil bacterial community through the extraction of microbial DNA from soil and generation of bacterial community profiles by PCR amplification of 16S rRNA genes. Extraction efficiency of soil microbial DNA is the most important step in these methods.

Zhou Xiaoqi; Wang Yanfen; Cai Ying; Huang Xiangzhong; Hao Yanbin; Tian Jianqing; Chai Tuanyao

2007-01-01

15

Comparison of Bacterial Extracellular Polymer Extraction Methods  

PubMed Central

Five different bacterial extracellular polymer extraction methods and a combination of two of these methods were compared on cultures of activated sludge, synthetic activated sludge, and Klebsiella aerogenes. High-speed centrifugation was the most effective extraction method for the K. aerogenes culture, based on the comparatively small amount of cell disruption and the relatively high extracellular polymer yield. Steaming treatment was the most effective extraction method for the activated sludges, since it released a significant quantity of extracellular polymers from the flocs and caused less cellular disruption than ethylenediaminetetraacetic acid and sodium hydroxide treatments. Sodium hydroxide treatment caused extensive disruption in all cultures. Ultrasonication released low concentrations of extracellular polymers from all cultures. However, it caused no significant cell disruption and therefore may be useful as a preliminary treatment in conjunction with another extraction method.

Brown, Melanie J.; Lester, John N.

1980-01-01

16

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.  

PubMed Central

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.

Krueger, D M; Cavanaugh, C M

1997-01-01

17

A Comparison of rpoB and 16S rRNA as Markers in Pyrosequencing Studies of Bacterial Diversity  

PubMed Central

Background The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. Methodology/Principal Findings Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. Conclusions/Significance The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.

Pijl, Agata S.; de Hollander, Mattias; Kowalchuk, George A.

2012-01-01

18

Analysis of the Cystic Fibrosis Lung Microbiota via Serial Illumina Sequencing of Bacterial 16S rRNA Hypervariable Regions  

PubMed Central

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

Diaz Caballero, Julio; Fung, Pauline; Gong, Yunchen; Donaldson, Sylva L.; Yuan, Lijie; Keshavjee, Shaf; Zhang, Yu; Yau, Yvonne C. W.; Waters, Valerie J.; Tullis, D. Elizabeth; Hwang, David M.; Guttman, David S.

2012-01-01

19

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

PubMed

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

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

2011-06-30

20

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

PubMed Central

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

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

2012-01-01

21

Occurrence of Fragmented 16S rRNA in an Obligate Bacterial Endosymbiont of Paramecium caudatum  

Microsoft Academic Search

The phylogenetic position of Caedibacter caryophila, a so far noncultured killer symbiont of Paramecium caudatum, was elucidated by comparative sequence analysis of in vitro amplified 16S rRNA genes (rDNA). C. caryophila is a member of the alpha subclass of the Proteobacteria phylum. Within this subclass C. caryophila is moderately related to Holospora obtusa, which is another obligate endosymbiont of Paramecium

Nina Springer; Wolfgang Ludwig; Rudolf Amann; Helmut Josef Schmidt; Hans-Dieter Gortz; Karl-Heinz Schleifer

1993-01-01

22

Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA  

PubMed Central

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

Marchesi, Julian R.; Sato, Takuichi; Weightman, Andrew J.; Martin, Tracey A.; Fry, John C.; Hiom, Sarah J.; Wade, William G.

1998-01-01

23

Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas ( Lama pacos) and sheep ( Ovis aries)  

Microsoft Academic Search

Two bacterial 16S rRNA gene clone libraries were constructed from the forestomach of alpacas and sheep fed alfalfa. After the amplification using the universal 16S rRNA gene primers, equal quantities of PCR products from the same species were mixed and used to construct the two libraries. Sequence analysis showed that the 60 clones from alpacas were divided into 27 phylotypes

Cai-Xia Pei; Qiang Liu; Chang-Sheng Dong; HongQuan Li; Jun-Bing Jiang; Wen-Jun Gao

2010-01-01

24

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

Microsoft Academic Search

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries.

BALBINA NOGALES; EDWARD R. B. MOORE; ENRIQUE LLOBET-BROSSA; RAMON ROSSELLO-MORA; RUDOLF AMANN; KENNETH N. TIMMIS

2001-01-01

25

Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge.  

PubMed

A culture-independent molecular technique using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes was applied to the characterization of bacterial communities of activated sludge taken from different municipal sewage treatment plants. 16S rDNA fragments from the bulk DNA of sludge were amplified by PCR with a Cy5-labeled forward primer corresponding to nucleotide positions 8 to 27 and a reverse primer complementary to positions 907 to 926 in the Escherichia coli numbering system. The 16S rDNAs thus obtained were digested with tetrameric restriction enzymes and analyzed using a Pharmacia automated DNA sequencer. A preliminary study on a model DNA mixture prepared from different bacterial species showed that the fluorescence intensity of terminal fragments (T-RFs) of 16S rDNAs amplified and detected was directly proportional to the 16S rRNA gene copy number rather than the amount of genomic DNA of each species present. 16S rDNA fragments amplified from the sludges and digested with HhaI usually generated at least 60 T-RFs, among which T-RFs of around 208 bp were the most abundant regardless of the time or area sampled. Southern blot hybridization with oligonucleotide probes specific to the 5' terminal regions of the 16S rDNA of different phylogenetic groups indicated that the T-RFs of around 208 bp were derived from members of the beta subclass of the class Proteobacteria. Hybridization with a probe specific to the class Actinobacteria failed to detect any appreciable signal. These results did not agree fully with those obtained by quinone profiling. The usefulness and limitations of the T-RFLP method for monitoring bacterial population dynamics in activated sludge were discussed. PMID:16232834

Hiraishi, A; Iwasaki, M; Shinjo, H

2000-01-01

26

Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies.  

PubMed

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies. PMID:22880076

Vasileiadis, Sotirios; Puglisi, Edoardo; Arena, Maria; Cappa, Fabrizio; Cocconcelli, Pier S; Trevisan, Marco

2012-08-06

27

Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.  

PubMed Central

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry.

Hiorns, W D; Methe, B A; Nierzwicki-Bauer, S A; Zehr, J P

1997-01-01

28

Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.  

PubMed

Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR. PMID:23139793

Clifford, Robert J; Milillo, Michael; Prestwood, Jackson; Quintero, Reyes; Zurawski, Daniel V; Kwak, Yoon I; Waterman, Paige E; Lesho, Emil P; Mc Gann, Patrick

2012-11-06

29

Macroalgal extracts induce bacterial assemblage shifts and sublethal tissue stress in Caribbean corals.  

PubMed

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways. PMID:23028648

Morrow, Kathleen M; Ritson-Williams, Raphael; Ross, Cliff; Liles, Mark R; Paul, Valerie J

2012-09-13

30

Macroalgal Extracts Induce Bacterial Assemblage Shifts and Sublethal Tissue Stress in Caribbean Corals  

PubMed Central

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways.

Morrow, Kathleen M.; Ritson-Williams, Raphael; Ross, Cliff; Liles, Mark R.; Paul, Valerie J.

2012-01-01

31

Response of a Soil Bacterial Community to Grassland Succession as Monitored by 16S rRNA Levels of the Predominant Ribotypes  

PubMed Central

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the ? subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.

Felske, Andreas; Wolterink, Arthur; Van Lis, Robert; De Vos, Willem M.; Akkermans, Antoon D. L.

2000-01-01

32

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities  

PubMed Central

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.

Dunbar, John; Ticknor, Lawrence O.; Kuske, Cheryl R.

2001-01-01

33

Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction--single-strand conformation polymorphism analysis.  

PubMed

The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a culture-independent approach based on single-strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the digester operating at steady state, the 16S rDNA SSCP patterns of the bacterial community showed eight peaks, whereas the 16S rRNA patterns showed six peaks with a very prominent one corresponding to a Spirochaetes-related bacterium. An acidic shock at pH 6 caused an increase in the 16S rRNA level of two Clostridium-related bacteria. After a 1 week starvation period, the major bacteria present reverted to a basal 16S rRNA level proportional to their 16S rDNA level. Starvation revealed the presence of a previously undetected peak whose corresponding sequence was deeply branched into the low G+C Gram-positive bacteria phylum. Twenty-four hours after a spiked addition to the starved digester community of starch, glucose, lactate or sulphate, an upsurge in several new 16S rRNA-derived peaks was observed. Thus, the perturbation approach combined with 16S rRNA analysis revealed bacteria that had not been detected through 16S rDNA analysis. PMID:11233159

Delbès, C; Moletta, R; Godon, J J

2000-10-01

34

Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. [Calyptogena magnifica; Bathymodiolus thermophilus; Lucinoma annulata; Lucinoma aequizonata; Codakia orbicularis  

SciTech Connect

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis. Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.

Distel, D.L.; Lane, D.J.; Olsen, G.J.; Giovannoni, S.J.; Pace, B.; Pace, N.R.; Stahl, D.A.; Felbeck, H.

1988-06-01

35

Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample  

SciTech Connect

Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the {gamma} subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the {gamma} subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases. 34 refs., 4 figs., 3 tabs.

Suzuki, M.T.; Rappe, M.S.; Haimberger, Z.W. [Oregon State Univ., Corvallis, OR (United States)] [and others

1997-03-01

36

Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample.  

PubMed Central

Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the gamma subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the gamma subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases.

Suzuki, M T; Rappe, M S; Haimberger, Z W; Winfield, H; Adair, N; Strobel, J; Giovannoni, S J

1997-01-01

37

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment  

PubMed Central

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions – provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.

Guo, Feng; Ju, Feng; Cai, Lin; Zhang, Tong

2013-01-01

38

Ultradeep 16S rRNA Sequencing Analysis of Geographically Similar but Diverse Unexplored Marine Samples Reveal Varied Bacterial Community Composition  

PubMed Central

Background Bacterial community composition in the marine environment differs from one geographical location to another. Reports that delineate the bacterial diversity of different marine samples from geographically similar location are limited. The present study aims to understand whether the bacterial community compositions from different marine samples harbour similar bacterial diversity since these are geographically related to each other. Methods and Principal Findings In the present study, 16S rRNA deep sequencing analysis targeting V3 region was performed using Illumina bar coded sequencing. A total of 22.44 million paired end reads were obtained from the metagenomic DNA of Marine sediment, Rhizosphere sediment, Seawater and the epibacterial DNA of Seaweed and Seagrass. Diversity index analysis revealed that Marine sediment has the highest bacterial diversity and the least bacterial diversity was observed in Rhizosphere sediment. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant taxa present in all the marine samples. Nearly 62–71% of rare species were identified in all the samples and most of these rare species were unique to a particular sample. Further taxonomic assignment at the phylum and genus level revealed that the bacterial community compositions differ among the samples. Conclusion This is the first report that supports the fact that, bacterial community composition is specific for specific samples irrespective of its similar geographical location. Existence of specific bacterial community for each sample may drive overall difference in bacterial structural composition of each sample. Further studies like whole metagenomic sequencing will throw more insights to the key stone players and its interconnecting metabolic pathways. In addition, this is one of the very few reports that depicts the unexplored bacterial diversity of marine samples (Marine sediment, Rhizosphere sediment, Seawater) and the host associated marine samples (Seaweed and Seagrass) at higher depths from uncharacterised coastal region of Palk Bay, India using next generation sequencing technology.

Karutha Pandian, Shunmugiah

2013-01-01

39

Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory  

PubMed Central

We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

BOOMER, SARAH M.; LODGE, DANIEL P.; DUTTON, BRYAN E.

2002-01-01

40

Two-Dimensional Combinatorial Screening (2DCS) of a Bacterial rRNA A-site-like Motif Library: Defining Privileged Asymmetric Internal Loops that Bind Aminoglycosides  

PubMed Central

RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the ribosomal RNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1×1 nucleotide all-U internal loop and a 2×1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using Two-Dimensional Combinatorial Screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6?-azido-kanamycin A, 5-O-(2-azidoethyl) neamine, and 6?-azido-tobramycin, the selected internal loops bind with ~10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5?-azido-neomycin B bind with similar affinity as the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they were selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that could be used to enable the rational and modular design of small molecules targeting RNA.

Tran, Tuan; Disney, Matthew D.

2010-01-01

41

Composition of the bacterial population of refrigerated beef, identified with direct 16S rRNA gene analysis and pure culture technique.  

PubMed

The composition of the dominating population of freshly cut beef, and beef stored at 4 degrees C for 8 d, was studied by direct analysis of the 16S rRNA gene (PCR amplification, cloning and sequencing) and compared with pure culture technique where the isolates picked from the viable plate count were identified by sequencing of the 16S rRNA gene. The composition of the bacterial population was recorded at two different time points, at the start when the viable plate count of the meat was 4 x 10(2) colony forming unit (cfu) per cm(2) and when it was 5 x 10(7) cfu per cm(2). Direct gene analysis by PCR amplification generated 30 clones, and 79 isolates were picked from the plate count, and identified by 16S rRNA gene sequencing. At the low initial bacterial load of the beef, the two sampling strategies showed variations in the composition of species. Direct 16S rRNA gene analysis revealed a domination of Bacillus-like sequences while no such sequences were found in isolates from the viable plate count. Instead the population of the plate count was dominated by Chryseobacterium spp. In contrast, the two sampling strategies matched on the multiplying beef population, where both methods indicated Pseudomonas spp. as the dominating group (99% of the population-sequences), irrespectively of sampling strategy. Pseudomonas panacis/Pseudomons brennerii was the dominating taxon (99% similarity to type strain), but sequences with highest similarity to Pseudomonas lundensis (99%), Pseudomonas beteli (99%) and Pseudomonas koreensis (100%) were also found. PMID:17765348

Olofsson, T C; Ahrné, S; Molin, G

2007-07-31

42

Bacterial community composition during two consecutive NE Monsoon periods in the Arabian Sea studied by denaturing gradient gel electrophoresis (DGGE) of rRNA genes  

NASA Astrophysics Data System (ADS)

Horizontal and vertical variations in bacterial community composition were examined in samples collected during two Joint Global Ocean Flux Study (JGOFS) Arabian Sea cruises in 1995. The cruises, 11 months apart, took place during two consecutive NE Monsoon periods (January and December). Bacteria were harvested by filtration from samples collected in the mixed layer, mid-water, and deep sea at stations across the study area. Total bacterial community genomic DNA was analyzed by PCR amplification of 16S rRNA gene fragments, followed by denaturing gradient gel electrophoresis (DGGE). In total, 20 DGGE bands reflecting unique or varying phylotypes were excised, cloned and sequenced. Amplicons were dominated by bacterial groups commonly found in oceanic waters (e.g., the SAR11 cluster of ?-Proteobacteria and cyanobacteria), but surprisingly none of the sequenced amplicons were related to ?-Proteobacteria or to members of the Cytophaga-Flavobacter-Bacteroides phylum. Amplicons related to magnetotactic bacteria were found for the first time in pelagic oceanic waters. The DGGE banding patterns revealed a dominance of ?15 distinguishable amplicons in all samples. In the mixed layer the bacterial community was dominated by the same ?15 phylotypes at all stations, but unique phylotypes were found with increasing depth. Except for cyanobacteria, comparison of the bacterial community composition in surface waters from January and December 1995 showed only minor differences, despite significant differences in environmental parameters. These data suggest a horizontal homogeneity and some degree of seasonal predictability of bacterial community composition in the Arabian Sea.

Riemann, Lasse; Steward, Grieg F.; Fandino, Laura B.; Campbell, Lisa; Landry, Michael R.; Azam, Farooq

1999-08-01

43

Rapid qualitative characterization of bacterial community in eutrophicated wastewater stabilization plant by T-RFLP method based on 16S rRNA genes.  

PubMed

Waste stabilization ponds are a simple, low-cost extensive process for treating wastewater, and well adapted to low socio-economic conditions in developing countries where the microbial populations in these systems are not well characterized. The phylogenetic bacterial community structure within a Tunisian wastewater stabilization plant treating domestic wastewater was assessed by Terminal Restriction Fragment Length Polymorphism method targeting 16S rRNA genes and by the APLAUS+ software of the Microbial Community Analysis (MiCA) web based tool. The dimeric enzymatic digestion with HaeIII and HinfI restriction enzymes revealed high bacterial diversity within the plant where 11 bacterial phyla were identified. The total bacterial community structure includes bacteria catalysing nitrogen and phosphorus removal and bacteria involved in the sulfur cycle. The bacterial community was characterized by the dominance of Proteobacteria which was the most populous phylum (60%) followed by the Actinobacteria (20%), the Firmicutes (10.3%), the Bacteroidetes (2.3%), the Nitrospira (2.2%). Minor bacterial phyla groups occupied smaller fractions such as Chloroflexi, Deferribacteres and Verrumicrobia. T-RFLP analysis revealed also that The Proteobacteria phylum was characterized by the dominance of bacteria of The Gammaproteobacteria class. PMID:22806789

Belila, Abdelaziz; Snoussi, Mejdi; Hassan, Abdennaceur

2011-06-10

44

Bacterial diversity from the source to the tap: a comparative study based on 16S rRNA gene-DGGE and culture-dependent methods.  

PubMed

This study aimed to assess the influence of water treatment and distribution on the bacterial communities with particular emphasis on tap water. Samples from the water treatment plant, the bulk supply distribution system and household taps, supplied by the same drinking water treatment plant, were analyzed using culture-dependent and culture-independent methods. Water treatment imposed alterations in the composition of the bacterial community, although this effect was more evident in the cultivable bacteria rather than among the total community assessed by 16S rRNA gene-denaturing gradient gel electrophoresis (DGGE) profiling. Water disinfection, mainly chlorination, promoted a reduction on bacterial diversity and cultivability, with a shift in the pattern of cultivable bacteria from predominantly Gram-negative to predominately Gram-positive and acid-fast. Downstream of the chlorination stages, tap water, in comparison with raw water, presented higher diversity indices and cultivability percentages. From the source to the tap, members of the Alpha-, Beta- and Gammaproteobacteria were the predominant lineages identified using 16S rRNA gene-DGGE analysis. Although with a lower coverage, the DGGE-based lineage identifications were in agreement with those found using 454-pyrosequencing analysis. Despite the effectiveness of water treatment to eliminate or inactivate most of the bacteria, Proteobacteria such as Acinetobacter, Bosea and Sphingomonadaceae may successfully colonize tap water. PMID:22938591

Vaz-Moreira, Ivone; Egas, Conceição; Nunes, Olga C; Manaia, Célia M

2012-09-17

45

Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA  

Microsoft Academic Search

The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal

BERNADETTE M. DUINEVELD; GEORGE A. KOWALCHUK; ANNEKE KEIJZER; JAN DIRK VAN ELSAS; JOHANNES A. VAN VEEN

2001-01-01

46

Comparison between MICRO-CARD-FISH and 16S rRNA gene clone libraries to assess the active versus total bacterial community in the coastal Arctic  

PubMed Central

We collected surface- and deep-water samples (maximum depth 300 m) during the spring–summer transition in the coastal Arctic along a transect in the Kongsfjorden (Ny-Ålesund, Spitsbergen, Norway) to determine the structure of the active versus total marine bacterioplankton community using different approaches. Catalysed reporter deposition–fluorescence in situ hybridization combined with microautoradiography (MICRO–CARD–FISH) was used to determine the abundance and activity of different bacterial groups. The bacterial communities were dominated by members of Alphaproteobacteria followed by Bacteroidetes, whereas Gammaproteobacteria were present at low abundance but exhibited a high percentage of active cells taking up leucine. The clone libraries of 16S rRNA genes (16S rDNA) and 16S rRNA from two different depths were used to decipher the bacterial community structure. Independently of the type of clone libraries analysed (16S rDNA- or 16S rRNA-based), four major and four minor taxonomic groups were detected. The bacterioplankton community was mainly dominated at both the DNA and the RNA levels by Alphaproteobacteria followed by Gammaproteobacteria. The Rhodobacteriaceae were the most abundant members of the Alphaproteobacteria in both DNA and RNA clone libraries, followed by the SAR11 clade, which was only detectable at the 16S rDNA level. Moreover, there was a general agreement between the results obtained with both techniques, although some specific phylogenetic groups, such as SAR11 and Roseobacter, deviated substantially from this relation. These discrepancies are most likely linked to different physiological states among members of the bacterioplankton community. Combined, MICRO–CARD–FISH and DNA and RNA clone libraries, however, allowed for accurately quantifying different bacterial groups and their activity as well as a detailed phylogenetic insight into the fractions of present versus metabolically active bacterial groups.

De Corte, Daniele; Sintes, Eva; Yokokawa, Taichi; Herndl, Gerhard J

2013-01-01

47

How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?  

PubMed Central

Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions.

Sergiev, Petr V.; Golovina, Anna Y.; Sergeeva, Olga V.; Osterman, Ilya A.; Nesterchuk, Mikhail V.; Bogdanov, Alexey A.; Dontsova, Olga A.

2012-01-01

48

Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample  

Microsoft Academic Search

Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not

MARCELINO T. SUZUKI; MICHAEL S. RAPPE; ZARA W. HAIMBERGER

1997-01-01

49

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

PubMed Central

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

2010-01-01

50

Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment  

Microsoft Academic Search

physiologic state of Acinetobacter calcoaceticus ATCC 23055 T. Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to

Ting Lu; Peter G. Stroot; Daniel B. Oerther

2009-01-01

51

Comprehensive phylogenetic analysis of evolutionarily conserved rRNA adenine dimethyltransferase suggests diverse bacterial contributions to the nucleus-encoded plastid proteome.  

PubMed

The KsgA/Dim1 protein family of rRNA adenine dimethyltransferase (rAD) is well conserved throughout evolution. This protein family has been recognized to play multiple additional roles: as a mitochondrial transcription factor (mtTFB); as a yeast pre-rRNA cleavage enzyme (Dim1p); and as a chloroplast developmental protein (PFC1). Comprehensive phylogenetic analysis of rAD led to three main findings. First, rAD sequences were grouped by three domains of life, bacteria, archaea, and eukaryotes. Second, mtTFB shows alpha-proteobacterial endosymbiotic ancestry. Third, plastid-targeted rADs do not show cyanobacterial affiliation. Instead, plastid-targeted homologs from chlorophytes/land plants were clustered with chlamydiae, while those from rhodophytes/red algal lineage grouped with the bacteroides/chlorobi group. These unusual two-bacterial ancestries of plastid-targeted rAD suggest that bacterial genes influenced the evolution of the proteome of eukaryotic plastids in a complex way, involving more diverse bacterial groups than previously believed, and underscoring the importance of eukaryotic acquisition of bacterial functions. PMID:19017544

Park, Ae Kyung; Kim, Ho; Jin, Hyung Jong

2008-11-05

52

Quantifying Microbial Utilization of Petroleum Hydrocarbons in Salt Marsh Sediments by Using the 13C Content of Bacterial rRNA?  

PubMed Central

Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we took a whole-community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon isotopic abundance between a salt marsh ecosystem supported by the 13C-enriched C4 grass Spartina alterniflora and 13C-depleted petroleum to monitor changes in the 13C content of biomass. Magnetic bead capture methods for selective recovery of bacterial RNA were used to monitor the 13C content of bacterial biomass during a 2-week experiment. The data show that by the end of the experiment, up to 26% of bacterial biomass was derived from consumption of the freshly spilled oil. The results contrast with the inertness of a nearby relict spill, which occurred in 1969 in West Falmouth, MA. Sequences of 16S rRNA genes from our experimental samples also were consistent with previous reports suggesting the importance of Gamma- and Deltaproteobacteria and Firmicutes in the remineralization of hydrocarbons. The magnetic bead capture approach makes it possible to quantify uptake of petroleum hydrocarbons by microbes in situ. Although employed here at the domain level, RNA capture procedures can be highly specific. The same strategy could be used with genus-level specificity, something which is not currently possible using the 13C content of biomarker lipids.

Pearson, Ann; Kraunz, Kimberly S.; Sessions, Alex L.; Dekas, Anne E.; Leavitt, William D.; Edwards, Katrina J.

2008-01-01

53

POLYMERASE CHAIN REACTION AMPLIFICATION OF BACTERIAL 16S rRNA GENES FROM COLD-CUP BIOPSY FORCEPS  

Microsoft Academic Search

PurposeIn looking for a possible infectious cause for interstitial cystitis (IC), we previously determined that bladder tissue specimens from both IC patients and controls were uniformly positive by polymerase chain reaction assay (PCR) for bacterial 16S ribosomal RNA genes from various genera including Escherichia, Propionobacterium, Acinetobacter, and Salmonella. We therefore determined whether the biopsy forceps might be contaminated with bacterial

S. KEAY; C. O. ZHANG; B. R. BALDWIN; R. B. ALEXANDER; J. W. WARREN

1998-01-01

54

Bacterial diversities on unaged and aging flue-cured tobacco leaves estimated by 16S rRNA sequence analysis  

Microsoft Academic Search

Flue-cured tobacco leaves (FCTL) contain abundant bacteria, and these bacteria play very important roles in the tobacco aging\\u000a process. However, bacterial communities on aging FCTL are not fully understood. In this study, the total microbial genome\\u000a DNA of unaged and aging flue-cured tobacco K326 were isolated using a culture-independent method, and the bacterial communities\\u000a were investigated by restriction fragment length

Jingwen Huang; Jinkui Yang; Yanqing Duan; Wen Gu; Xiaowei Gong; Wei Zhe; Can Su; Ke-Qin Zhang

2010-01-01

55

Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray  

Microsoft Academic Search

We have developed an oligonucleotide microarray for the detection of biodegradative genes and bacterial diversity and tested\\u000a it in five contaminated ecosystems. The array has 60-mer oligonucleotide probes comprising 14,327 unique probes derived from\\u000a 1,057 biodegradative genes and 880 probes representing 110 phylogenetic genes from diverse bacterial communities, and we named\\u000a it as BiodegPhyloChip. The biodegradative genes are involved in

Ashutosh Pathak; Rishi Shanker; Satyendra Kumar Garg; Natesan Manickam

2011-01-01

56

Bacterial Etiology for Chronic Villitis Is Not Supported by Polymerase Chain Reaction for 16S rRNA DNA  

Microsoft Academic Search

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture.

Linda M. Ernst; Jill Crouch; Henry Rinder; John Greg Howe

2005-01-01

57

Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave, Spain, and on its Palaeolithic paintings.  

PubMed

Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000-20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1-2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga-Flexibacter- Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments. PMID:12123475

Schabereiter-Gurtner, Claudia; Saiz-Jimenez, Cesareo; Piñar, Guadalupe; Lubitz, Werner; Rölleke, Sabine

2002-07-01

58

Small-subunit rRNA genes and in situ hybridization with oligonucleotides specific for the bacterial symbionts in the larvae of the bryozoan Bugula neritina and proposal of "Candidatus endobugula sertula".  

PubMed Central

Larvae of the bryozoan Bugula neritina harbor bacterial symbionts. These symbionts were identified as a novel species of gamma-proteobacterium, based on ribosomal small-subunit rRNA gene sequences. In situ hybridization with oligonucleotides specific for the symbiont confirmed the origin of the sequence. The taxonomic status "Candidatus Endobugula sertula" is proposed for the larval symbiont.

Haygood, M G; Davidson, S K

1997-01-01

59

Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.  

PubMed

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (?360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use. PMID:23734239

Salipante, Stephen J; Sengupta, Dhruba J; Rosenthal, Christopher; Costa, Gina; Spangler, Jessica; Sims, Elizabeth H; Jacobs, Michael A; Miller, Samuel I; Hoogestraat, Daniel R; Cookson, Brad T; McCoy, Connor; Matsen, Frederick A; Shendure, Jay; Lee, Clarence C; Harkins, Timothy T; Hoffman, Noah G

2013-05-29

60

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis.  

PubMed

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs. PMID:17115448

Paungfoo, Chanyarat; Prasertsan, Poonsuk; Burrell, Paul C; Intrasungkha, Nugul; Blackall, Linda L

2007-07-01

61

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

PubMed Central

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

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

2012-01-01

62

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel.  

PubMed

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control. PMID:22500778

Marty, Florence; Ghiglione, Jean-François; Païssé, Sandrine; Gueuné, Hervé; Quillet, Laurent; van Loosdrecht, Mark C M; Muyzer, Gerard

2012-01-01

63

Bacterial diversity in the rumen of Gayals (Bos frontalis), Swamp buffaloes (Bubalus bubalis) and Holstein cow as revealed by cloned 16S rRNA gene sequences.  

PubMed

Libraries of rumen bacterial 16S rRNA gene sequences of Gayals (Bos frontalis) and Swamp buffaloes (Bubalus bubalis) were cloned and sequenced in the present work to compare the bacterial diversity with the third published library of Holstein cow. Sequence similarity of 97% was used as the definition of operational taxonomic unit (OTU). The majority of the 470 sequences retrieved fell into the phyla of low G + C subdivision (329 sequences) and Cytophaga-Flexibacter-Bacteroides (CFB, 123 sequences) with the percentages of 70 and 26.2, respectively. The remaining clones belonged to the phyla of Proteobacter, high G + C gram positive bacteria (HGCGPB) and Spirochaetes, accounting for 3.8% totally. Only 73 clones (25 OTUs, 15.5%) could be closely related to cultured representatives. However, a larger fraction was related to uncultured representatives. Holstein cow may have more representatives of cultural bacteria and there were more uncultured clones for Gayals. The percentage of cultural representatives was 24, 13.3 and 9.5 for Holstein cow, Swamp buffaloes and Gayals, respectively. Twenty-three OTUs of the 236 ones appeared in more than one library, five of which were cultural. Selenomonas ruminantium, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens were found in two different libraries, while Succiniclasticum ruminis and Pseudobutyrivibrio ruminis were found in all three libraries. Some of the animal-specific bacteria that had not been described previously in the ruminal ecosystem, e.g. Allisonella histaminiformans for Gayals and Staphylococcus sciuri for Swamp buffaloes were also recovered. PMID:19662514

Yang, Shuli; Ma, Songcheng; Chen, Jing; Mao, Huaming; He, Yiduo; Xi, Dongmei; Yang, Liangyu; He, Tianbao; Deng, Weidong

2009-08-07

64

Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of Community Fingerprints  

PubMed Central

Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5? exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

Heuer, Holger; Hartung, Kathrin; Wieland, Gabriele; Kramer, Ina; Smalla, Kornelia

1999-01-01

65

Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers  

PubMed Central

Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.

McGarvey, Jeffery A.; Miller, William G.; Sanchez, Susan; Stanker, Larry

2004-01-01

66

Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection.  

PubMed

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

Lindsay, Brianna; Pop, Mihai; Antonio, Martin; Walker, Alan W; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Li, Shan; Magder, Laurence S; Paulson, Joseph N; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E; Hannis, James; Manalili, Sheri; Deleon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J; Hossain, M Anowar; Breiman, Robert F; Morris, J Glenn; McDaniel, Timothy K; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O Colin; Nataro, James P

2013-07-24

67

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

PubMed Central

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

Bolhuis, Henk; Stal, Lucas J

2011-01-01

68

Effects of season and experimental warming on the bacterial community in a temperate mountain forest soil assessed by 16S rRNA gene pyrosequencing  

PubMed Central

Climate warming may induce shifts in soil microbial communities possibly altering the long-term carbon mineralization potential of soils. We assessed the response of the bacterial community in a forest soil to experimental soil warming (+4 °C) in the context of seasonal fluctuations. Three experimental plots were sampled in the fourth year of warming in summer and winter and compared to control plots by 16S rRNA gene pyrosequencing. We sequenced 17 308 amplicons per sample and analysed operational taxonomic units at genetic distances of 0.03, 0.10 and 0.25, with respective Good's coverages of 0.900, 0.977 and 0.998. Diversity indices did not differ between summer, winter, control or warmed samples. Summer and winter samples differed in community structure at a genetic distance of 0.25, corresponding approximately to phylum level. This was mainly because of an increase of Actinobacteria in winter. Abundance patterns of dominant taxa (> 0.06% of all reads) were analysed individually and revealed, that seasonal shifts were coherent among related phylogenetic groups. Seasonal community dynamics were subtle compared to the dynamics of soil respiration. Despite a pronounced respiration response to soil warming, we did not detect warming effects on community structure or composition. Fine-scale shifts may have been concealed by the considerable spatial variation.

Kuffner, Melanie; Hai, Brigitte; Rattei, Thomas; Melodelima, Christelle; Schloter, Michael; Zechmeister-Boltenstern, Sophie; Jandl, Robert; Schindlbacher, Andreas; Sessitsch, Angela

2012-01-01

69

Improved soil dispersion procedures for total bacterial counts, extraction of indigenous bacteria and cell survival  

Microsoft Academic Search

The effects of different soil dispersion procedures for enumeration and extraction of indigenous soil bacteria were investigated. Increased counts and extraction efficiency were obtained with pyrophosphate instead of water as dispersion liquid. When physical dispersion was conducted in the Waring blender, the effect of container volume and number of dispersions on extraction efficiency and bacterial counts was shown. An extraction

Viggo Lindahl

1996-01-01

70

Anti-Acne-Inducing Bacterial Activity of Mangosteen Fruit Rind Extracts  

Microsoft Academic Search

Objective: The aims of this study were to determine the most effective solvent extract of mangosteen, anti-acne- inducing bacterial activity and the amount of ?-mangostin, a major active component in each mangosteen fruit rind extract, using high-performance liquid chromatography (HPLC). Materials and Methods: The fruit rinds of mangosteen were extracted with hexane, dichloromethane, ethanol and water. The extracts were tested

Werayut Pothitirat; Mullika Traidej Chomnawang; Wandee Gritsanapan

2010-01-01

71

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil  

SciTech Connect

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

2009-09-01

72

Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars  

Microsoft Academic Search

Direct genomic DNA extraction from vinegars was set up and suitability for PCR assays performed by PCR\\/DGGE and sequencing of 16S rRNA gene. The method was tested on 12 intermediary products of special vinegars, fruit vinegars and condiments produced from different raw materials and procedures. DNAs extraction was performed on pellets by chemical, enzymatic, resin mediated methods and their modifications.

Dhouha Mamlouk; Claudio Hidalgo; María-Jesús Torija; Maria Gullo

2011-01-01

73

A distinctive epibiotic bacterial community on the soft coral Dendronephthya sp. and antibacterial activity of coral tissue extracts suggest a chemical mechanism against bacterial epibiosis.  

PubMed

Abstract Different bacterial community profiles were observed on the soft coral Dendronephthya sp. and an inanimate reference site using terminal restriction fragment length polymorphism analysis of bacterial community DNA. To correlate the observation with a chemical defense mechanism against bacterial epibiosis, antibacterial effects of coral tissue extracts and waterborne products of coral-associated bacterial isolates (11 morphotypes) were tested against indigenous benthic bacterial isolates (33 morphotypes) obtained in the vicinity of the coral colonies. The coral tissue extracts and waterborne products of coral-associated bacteria inhibited growth and attachment of indigenous bacterial isolates, suggesting an endogenous chemical and an exogenous biological mechanism against bacterial epibiosis in this soft coral. PMID:19719665

Harder, Tilmann; Lau, Stanley C K; Dobretsov, Sergey; Fang, Tsz K; Qian, Pei-Yuan

2003-04-01

74

Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay  

PubMed Central

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5?-triphosphate (ATP), adenosine 5?-diphosphate (ADP), and adenosine 5?-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.

Lundin, Arne; Thore, Anders

1975-01-01

75

Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells.  

PubMed

Bacteria oxidize organic matter and nutrients to produce electric energy in microbial fuel cells (MFC) - a technology of increasing importance because of its sustainability. To improve the performance of MFCs, it is necessary not only to gain a better understanding of MFC engineering designs, but also to improve the understanding of the composition of the microbial communities in MFCs. Fast and efficient DNA extraction protocols that are suitable for extracting diverse bacterial genomes are necessary to identify the bacterial diversity present in MFCs and to further monitor the dynamic changes of microbial communities. This study focused on testing different direct cell lysis protocols to extract DNA from a microbial sludge harvested from an MFC. The protocol that achieved the best results was based on a previous study, but was modified by eliminating a chaotropic salt and the special columns used for nucleic acid purification. The efficiency of this less expensive and more straightforward protocol was confirmed by PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis analysis, which confirmed the extraction of multiple genomes. The sequences of 10 clones revealed the presence of phyla, Proteobacteria, Firmicutes and Actinobacteria, comprising both Gram-negative and Gram-positive bacteria. Some of these bacteria were identified at the genus level, e.g., Clostridium, Pseudoxanthomonas, Tistrella, and Enterobacter; these genera have been described in active sludges from wastewater treatment, supporting the congruency of our results. Therefore, this protocol is a useful tool for analysis of the bacteria responsible for energy production in MFCs. PMID:23408415

Canto-Canché, B; Tzec-Simá, M; Vázquez-Loría, J I; Espadas-Álvarez, H; Chí-Manzanero, B H; Rojas-Herrera, R; Valdez-Ojeda, R; Alzate-Gaviria, L

2013-02-04

76

Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes  

Microsoft Academic Search

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from\\u000a a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S\\u000a rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR\\u000a to optimize

Farida Hilali; Patrick Saulnier; Elisabeth Chachaty; Antoine Andremont

1997-01-01

77

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

PubMed Central

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

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

2002-01-01

78

Soil bacterial community structure in five tropical forests in Malaysia and one temperate forest in Japan revealed by pyrosequencing analyses of 16S rRNA gene sequence variation.  

PubMed

Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40-50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40-60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro- and macro-environments, but macro-environmental diversity made a larger contribution to total bacterial diversity in forest soil. PMID:23832301

Miyashita, Naohiko T; Iwanaga, Hiroko; Charles, Suliana; Diway, Bibian; Sabang, John; Chong, Lucy

2013-01-01

79

Dynamics and rRNA transcriptional activity of lactococci and lactobacilli during Cheddar cheese ripening.  

PubMed

Cheddar cheese is a complex ecosystem where both the bacterial population and the cheese making process contribute to flavor and texture development. The aim of this study was to use molecular methods to evaluate the impact of milk heat treatment and ripening temperature on starter lactococci and non-starter lactic acid bacteria (NSLAB) throughout ripening of Cheddar cheese. Eight Cheddar cheese batches were manufactured (four with thermized and four with pasteurized milk) and ripened at 4, 7 and 12°C to analyze the bacterial composition and rRNA transcriptional activity reflecting the ability of lactococci and lactobacilli to synthesize proteins. Abundance and rRNA transcription of lactococci and lactobacilli were quantified after DNA and RNA extraction by using quantitative PCR (qPCR) and reverse transcription-quantitative PCR (RT-qPCR) targeting the 16S rRNA gene, respectively. Results showed that lactococci remained dominant throughout ripening, although 16S rRNA genome and cDNA copies/g of cheese decreased by four and two log copy numbers, respectively. Abundance and rRNA transcription of Lactobacillus paracasei, Lactobacillus buchneri/parabuchneri, Lactobacillus rhamnosus, Lactobacillus brevis, and Lactobacillus coryniformis as well as total lactobacilli were also estimated using specific 16S rRNA primers. L. paracasei and L. buchneri/parabuchneri concomitantly grew in cheese made from thermized milk at 7 and 12°C, although L. paracasei displayed the most rRNA transcription among Lactobacillus species. This work showed that rRNA transcriptional activity of lactococci decreased throughout ripening and supports the usefulness of RNA analysis to assess which bacterial species have the ability to synthesize proteins during ripening, and could thereby contribute to cheese quality. PMID:23850855

Desfossés-Foucault, Emilie; Lapointe, Gisèle; Roy, Denis

2013-06-26

80

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities  

Microsoft Academic Search

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the

JOHN DUNBAR; LAWRENCE O. TICKNOR; CHERYL R. KUSKE

2001-01-01

81

Frequency of Formation of Chimeric Molecules as a Consequence of PCR Coamplification of 16S rRNA Genes from Mixed Bacterial Genomes  

Microsoft Academic Search

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of

GRACE C.-Y. WANG; YUE WANG

1997-01-01

82

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library.  

PubMed

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation. PMID:20559826

Garcia-Armisen, Tamara; Papalexandratou, Zoi; Hendryckx, Hugo; Camu, Nicholas; Vrancken, Gino; De Vuyst, Luc; Cornelis, Pierre

2010-06-18

83

The green alga Dicytosphaeria ocellata and its organic extracts alter natural bacterial biofilm communities.  

PubMed

Surfaces immersed in the marine environment are under intense fouling pressure by a number of invertebrates and algae. The regulation of this fouling can often be attributed to the bacterial biofilm that quickly develops on the surface of any available substratum in the ocean. The bacterial community composition on the surface of the green alga Dictyosphaeria ocellata was investigated and compared to those found on two other green algae, Batophora oerstedii and Cladophoropsis macromeres, and on a reference surface from three sites along the Florida Keys. Although the bacterial community composition of D. ocellata was not consistent across the sites, it was significantly different from the other algae and the reference surface at two of the three sites tested. Methanol extracts of D. ocellata significantly affected the abundance of bacteria and composition of the bacterial community on Phytagel™ plates when compared to solvent controls, suggesting that the alga regulates the bacterial community by producing active metabolites. PMID:21512919

Sneed, Jennifer M; Pohnert, Georg

2011-04-01

84

Comprehensive phylogenetic analysis of evolutionarily conserved rRNA adenine dimethyltransferase suggests diverse bacterial contributions to the nucleus-encoded plastid proteome  

Microsoft Academic Search

The KsgA\\/Dim1 protein family of rRNA adenine dimethyltransferase (rAD) is well conserved throughout evolution. This protein family has been recognized to play multiple additional roles: as a mitochondrial transcription factor (mtTFB); as a yeast pre-rRNA cleavage enzyme (Dim1p); and as a chloroplast developmental protein (PFC1). Comprehensive phylogenetic analysis of rAD led to three main findings. First, rAD sequences were grouped

Ae Kyung Park; Ho Kim; Hyung Jong Jin

2009-01-01

85

Supercritical fluid extraction of bacterial and archaeal lipid biomarkers from anaerobically digested sludge.  

PubMed

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile. PMID:22489140

Hanif, Muhammad; Atsuta, Yoichi; Fujie, Koichi; Daimon, Hiroyuki

2012-03-06

86

Supercritical Fluid Extraction of Bacterial and Archaeal Lipid Biomarkers from Anaerobically Digested Sludge  

PubMed Central

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile.

Hanif, Muhammad; Atsuta, Yoichi; Fujie, Koichi; Daimon, Hiroyuki

2012-01-01

87

Microfluidic device for bacterial genome extraction and analysis  

NASA Astrophysics Data System (ADS)

Although single molecule DNA manipulation and analysis techniques are emerging, methods for whole genome extraction from single cells, genomic length DNA handling and analytics is still to be developed. Here we present a microfabricated device to address some of these needs. This microfluidic chip is suitable for culturing bacteria and subsequently retrieve their genetic content. As a next step, the extracted DNA can be introduced in a nanostructured segment of the chip for precise handling, stretching and analysis. We hope that similar microdevices can be useful in studying genetic aspects of the cell lifecycle in a variety of organisms.

Galajda, Peter; Riehn, Robert; Wang, Yan-Mei; Keymer, Juan; Golding, Ido; Cox, Edward C.; Austin, Robert H.

2006-03-01

88

Antibacterial properties of extracts from selected planktonic freshwater cyanobacteria--a comparative study of bacterial bioassays.  

PubMed

Aqueous and methanol extracts from five selected cyanobacteria were examined for antibacterial properties in six different bacterial bioassays. All five cyanobacteria revealed antibacterial properties. Methanol extracts made from Tychonema bourrellyi, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii showed the most pronounced inhibitory effects, Aqueous extracts made from Microcystis aeruginosa and T. bourrellyi possessed evident antibacterial properties. The bacterial bioassays were based on agar diffusion tests and included pour-plate methods commonly used to detect residues of antibacterial substances in food. In addition, a pourplate bioassay with Aeromonas hydrophila was developed and described. Antibacterial effects were observed in five of the six bacterial bioassays. No antibacterial effect was observed in the Micrococcus luteus bioassay. Bioassays based on Aer. hydrophila, Bacillus cereus and B. subtilis grown in Antibiotic Medium 8, pH5.85, seemed to be sensitive and suitable. The MIC value of diluted MeOH extracts made from C. raciborskii and T. bourrellyi against Aer. hydrophila corresponded to 38 mg freeze-dried cyanobacteria. Bacillus subtilis was more sensitive when grown in a culture medium with pH 5.85 than 7.9. The antibacterial properties of extracts from the cyanobacteria examined differed from defined cyanotoxins and antibacterial substances. The pattern of inhibition in the bacterial bioassays indicated that various antibacterial substances are involved. PMID:9717298

Ostensvik, O; Skulberg, O M; Underdal, B; Hormazabal, V

1998-06-01

89

Bacterial Primary Colonization and Early Succession on Surfaces in Marine Waters as Determined by Amplified rRNA Gene Restriction Analysis and Sequence Analysis of 16S rRNA Genes  

PubMed Central

The nearly universal colonization of surfaces in marine waters by bacteria and the formation of biofilms and biofouling communities have important implications for ecological function and industrial processes. However, the dynamics of surface attachment and colonization in situ, particularly during the early stages of biofilm establishment, are not well understood. Experimental surfaces that differed in their degrees of hydrophilicity or hydrophobicity were incubated in a salt marsh estuary tidal creek for 24 or 72 h. The organisms colonizing these surfaces were examined by using a cultivation-independent approach, amplified ribosomal DNA restriction analysis. The goals of this study were to assess the diversity of bacterial colonists involved in early succession on a variety of surfaces and to determine the phylogenetic affiliations of the most common early colonists. Substantial differences in the representation of different cloned ribosomal DNA sequences were found when the 24- and 72-h incubations were compared, indicating that some new organisms were recruited and some other organisms were lost. Phylogenetic analyses of the most common sequences recovered showed that the colonists were related to organisms known to inhabit surfaces or particles in marine systems. A total of 22 of the 26 clones sequenced were affiliated with the Roseobacter subgroup of the ? subdivision of the division Proteobacteria (?-Proteobacteria), and most of these clones were recovered at a high frequency from all surfaces after 24 or 72 h of incubation. Two clones were affiliated with the Alteromonas group of the ?-Proteobacteria and appeared to be involved only in the very early stages of colonization (within the first 24 h). A comparison of the colonization patterns on the test surfaces indicated that the early bacterial community succession rate and/or direction may be influenced by surface physicochemical properties. However, organisms belonging to the Roseobacter subgroup are ubiquitous and rapid colonizers of surfaces in coastal environments.

Dang, Hongyue; Lovell, Charles R.

2000-01-01

90

Bacterial Communities within Digestive Tracts of Ground Beetles (Coleoptera: Carabidae)  

Microsoft Academic Search

We identiÞed the bacterial communities within the alimentary tracts of two granivorous ground beetles as a Þrst step in the exploration of bacteriaÐ ground beetle symbioses. Terminal- restriction fragment length polymorphism analyses of bacterial rRNA extracted from the guts of Þeld-collected individuals of Harpalus pensylvanicus (DeGeer) and Anisodactylus sanctaecrucis (F.) (Coleoptera: Carabidae) revealed that gut-associated bacterial communities were of low

Jonathan G. Lundgren; R. Michael Lehman; Joanne Chee-sanford

2007-01-01

91

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples  

PubMed Central

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Smith, K.; Diggle, M. A.; Clarke, S. C.

2003-01-01

92

The Inhibiting Effect of Aqueous Azadirachta indica (Neem) Extract Upon Bacterial Properties Influencing in vitro Plaque Formation  

Microsoft Academic Search

The purpose of this investigation was to examine the inhibitory effects of aqueous extracts derived from the bark-containing sticks (Neem stick) of Azadirachta indica upon bacterial aggregation, growth, adhesion to hydroxyapatite, and production of insoluble glucan, which may affect in vitro plaque formation. Neem stick extracts were screened for minimal bacterial growth inhibition (MIC) against a panel of streptococci by

L. E. Wolinsky; S. Mania; S. Nachnani; S. Ling

1996-01-01

93

Changes of the bacterial assemblages throughout an urban drinking water distribution system  

Microsoft Academic Search

We analyzed the bacterial 16S rRNA gene diversity throughout the major components of the drinking water distribution system\\u000a of a ca. 52,000-inhabitants city (Trikala City, Greece) in order to describe the changes of the bacterial assemblages and\\u000a to detect possible bacterial pathogens which are not included in the standard monitoring process. Bacterial DAPI counts and\\u000a DNA extraction was performed in

Konstantinos Ar. Kormas; Christos Neofitou; Maria Pachiadaki; Eulalia Koufostathi

2010-01-01

94

Bacterial RNA extraction and purification from whole human blood using isotachophoresis.  

PubMed

We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida . This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR. PMID:22816776

Rogacs, Anita; Qu, Yatian; Santiago, Juan G

2012-06-29

95

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification  

Microsoft Academic Search

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial\\u000a cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial\\u000a identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent

Seetharaman Vaidyanathan; Douglas B. Kell; Royston Goodacre

2002-01-01

96

Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.  

PubMed Central

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity. Images

Moyer, C L; Dobbs, F C; Karl, D M

1994-01-01

97

Diverse and novel oral bacterial species in blood following dental procedures.  

PubMed

We identified oral bacterial species in blood cultures following single-tooth extraction and tooth brushing. Sequence analysis of 16S rRNA genes identified 98 different bacterial species recovered from 151 bacteremic subjects. Of interest, 48 of the isolates represented 19 novel species of Prevotella, Fusobacterium, Streptococcus, Actinomyces, Capnocytophaga, Selenomonas, and Veillonella. PMID:18434561

Bahrani-Mougeot, Farah K; Paster, Bruce J; Coleman, Shirley; Ashar, Jignya; Barbuto, Sara; Lockhart, Peter B

2008-04-23

98

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

Microsoft Academic Search

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially

Henk Bolhuis; Lucas J Stal

2011-01-01

99

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

Microsoft Academic Search

BACKGROUND: Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering

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

2010-01-01

100

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

Microsoft Academic Search

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition

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

2003-01-01

101

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

Microsoft Academic Search

Received 19 January 1999\\/Returned for modification 2 May 1999\\/Accepted 26 June 1999 In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture

MICHAEL M. TUNNEY; SHEILA PATRICK; MARTIN D. CURRAN; GORDON RAMAGE; DONNA HANNA; JAMES R. NIXON; SEAN P. GORMAN; RICHARD I. DAVIS; NEIL ANDERSON

1999-01-01

102

Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts  

Microsoft Academic Search

Direct and sensitive PCR detection of contaminant microflora in cocoa extracts is affected by the quality of the template\\u000a DNA. This study compares the efficacy of five different commercial DNA extraction methods, selective enrichment broths and\\u000a use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artificially inoculated cocoa\\u000a extract samples. PCR-based methods

Marta Tortajada; Pedro Vicente Martínez-Culebras; Verónica Navarro; Honorato Monzó; Daniel Ramón

2009-01-01

103

Antimicrobial potential of Ricinus communis leaf extracts in different solvents against pathogenic bacterial and fungal strains  

PubMed Central

Objective To investigate the in vitro antimicrobial activities of the leaf extract in different solvents viz., methanol, ethanol and water extracts of the selected plant Ricinus communis. Methods Agar well diffusion method and agar tube dilution method were carried out to perform the antibacterial and antifungal activity of methanol, ethanol and aqueous extracts. Results Methanol leaf extracts were found to be more active against Gram positive bacteria (Bacillus subtilis: ATCC 6059 and Staphylococcus aureus: ATCC 6538) as well as Gram negative bacteria (Pseudomonas aeruginosa: ATCC 7221 and Klebsiella pneumoniae) than ethanol and aqueous leaf extracts. Antifungal activity of methanol and aqueous leaf extracts were also carried out against selected fungal strains as Aspergillus fumigatus and Aspergillus flavus. Methanolic as well as aqueous leaf extracts of Ricinus communis were effective in inhibiting the fungal growth. Conclusions The efficient antibacterial and antifungal activity of Ricinus communis from the present investigation revealed that the methanol leaf extracts of the selected plant have significant potential to inhibit the growth of pathogenic bacterial and fungal strains than ethanol and aqueous leaf extracts.

Naz, Rabia; Bano, Asghari

2012-01-01

104

DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer.  

PubMed

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow enhanced extraction of DNA from fluid suspensions. Characterization of the synthesis revealed linear doubling of the surface amine charge from generations one through five starting with an amino silane initiator. Furthermore, transmission electron microscopy revealed clear dispersion of the single domain magnetite in aqueous solution. The dendrimer modified magnetic particles have been used to carry out magnetic separation of DNA. Binding and release efficiencies increased with the number of generations and those of bacterial magnetite modified with six generation dendrimer were 7 and 11 times respectively as many as those of bacterial magnetite modified with only amino silane. PMID:12615391

Yoza, Brandon; Arakaki, Atsushi; Matsunaga, Tadashi

2003-03-20

105

Quantifying Microbial Utilization of Petroleum Hydrocarbons in Salt Marsh Sediments by Using the 13C Content of Bacterial rRNA  

Microsoft Academic Search

Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we took a whole- community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon isotopic abundance between a salt marsh ecosystem supported by the 13C-enriched C4 grass Spartina alterniflora and 13 C-depleted petroleum to monitor changes in

Ann Pearson; Kimberly S. Kraunz; Alex L. Sessions; Anne E. Dekas; William D. Leavitt; Katrina J. Edwards

2008-01-01

106

Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing  

Microsoft Academic Search

The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that

Uma Shankar Sagaram; Kristen M. DeAngelis; Pankaj Trivedi; Gary L. Andersen; Shi-En Lu; Nian Wang

2009-01-01

107

Diversity and composition of the bacterial community of Poyang Lake (China) as determined by 16S rRNA gene sequence analysis  

Microsoft Academic Search

Poyang Lake is the largest fresh water lake in China. In this study, the objective was to examine the diversity of bacterial\\u000a community in this environment. The phylogenetic composition of bacterioplankton communities from two sites and two dates (northern\\u000a and southern sub-basins in October 2006 and in May 2007, respectively) in the water column of Poyang Lake were investigated\\u000a by

Lan WuGang; Gang Ge; Guofeng Zhu; Shijie Gong; Siguang Li; Jinbao Wan

108

Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture-independent 16S rRNA techniques.  

PubMed

Culture-independent molecular (16S ribosomal RNA) techniques showed distinct differences in bacterial communities associated with white band disease (WBD) Type I and healthy elkhorn coral Acropora palmata. Differences were apparent at all levels, with a greater diversity present in tissues of diseased colonies. The bacterial community associated with remote, non-diseased coral was distinct from the apparently healthy tissues of infected corals several cm from the disease lesion. This demonstrates a whole-organism effect from what appears to be a localised disease lesion, an effect that has also been recently demonstrated in white plague-like disease in star coral Montastraea annularis. The pattern of bacterial community structure changes was similar to that recently demonstrated for white plague-like disease and black band disease. Some of the changes are likely to be explained by the colonisation of dead and degrading tissues by a micro-heterotroph community adapted to the decomposition of coral tissues. However, specific ribosomal types that are absent from healthy tissues appear consistently in all samples of each of the diseases. These ribotypes are closely related members of a group of alpha-proteobacteria that cause disease, notably juvenile oyster disease, in other marine organisms. It is clearly important that members of this group are isolated for challenge experiments to determine their role in the diseases. PMID:16703769

Pantos, Olga; Bythell, John C

2006-03-23

109

Diversity and composition of the bacterial community of Poyang Lake (China) as determined by 16S rRNA gene sequence analysis.  

PubMed

Poyang Lake is the largest fresh water lake in China. In this study, the objective was to examine the diversity of bacterial community in this environment. The phylogenetic composition of bacterioplankton communities from two sites and two dates (northern and southern sub-basins in October 2006 and in May 2007, respectively) in the water column of Poyang Lake were investigated by partially sequencing cloned 16SrRNA genes. Moreover, restriction fragment length polymorphism (RFLP) was applied in the 16SrRNA gene clones. In total, four clone libraries were constructed and 347 clones were screened by RFLP, yielding 153 operational taxonomic units, which mainly belonged to the proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Verrucomicrobia and Planctomycetes. Our results showed that Beta-proteobacteria was the most significant lineage, with dominant numbers of operational taxonomic units in the northern October 2006, southern October 2006 and May 2007 libraries. The highest bacterial diversity occurred in the library from the southern sub-basin in May 2007 and the lowest in the library from the northern sub-basin in May 2007. Horizontal and temporal differences associated with the concentration of total phosphorus, water temperature and pH suggested that the trophic state and the physicochemical properties of lake play key roles in sustaining bacterial community composition structure. PMID:22806799

Wu, Lan; Ge, Gang; Zhu, Guofeng; Gong, Shijie; Li, Siguang; Wan, Jinbao

2011-06-21

110

Identification of Clinical Coryneform Bacterial Isolates: Comparison of Biochemical Methods and Sequence Analysis of 16S rRNA and rpoB Genes?  

PubMed Central

We compared the relative levels of effectiveness of three commercial identification kits and three nucleic acid amplification tests for the identification of coryneform bacteria by testing 50 diverse isolates, including 12 well-characterized control strains and 38 organisms obtained from pediatric oncology patients at our institution. Between 33.3 and 75.0% of control strains were correctly identified to the species level by phenotypic systems or nucleic acid amplification assays. The most sensitive tests were the API Coryne system and amplification and sequencing of the 16S rRNA gene using primers optimized for coryneform bacteria, which correctly identified 9 of 12 control isolates to the species level, and all strains with a high-confidence call were correctly identified. Organisms not correctly identified were species not included in the test kit databases or not producing a pattern of reactions included in kit databases or which could not be differentiated among several genospecies based on reaction patterns. Nucleic acid amplification assays had limited abilities to identify some bacteria to the species level, and comparison of sequence homologies was complicated by the inclusion of allele sequences obtained from uncultivated and uncharacterized strains in databases. The utility of rpoB genotyping was limited by the small number of representative gene sequences that are currently available for comparison. The correlation between identifications produced by different classification systems was poor, particularly for clinical isolates.

Adderson, Elisabeth E.; Boudreaux, Jan W.; Cummings, Jessica R.; Pounds, Stanley; Wilson, Deborah A.; Procop, Gary W.; Hayden, Randall T.

2008-01-01

111

Bacterial cell wall components as immunomodulators—II. the bacterial cell wall extract OM85 BV as unspecific activator, immunogen and adjuvant in mice  

Microsoft Academic Search

The bacterial extract Broncho-Vaxom used for the prevention and treatment of recurrent respiratory tract infections is an immunomodulator in vitro and in vivo, as determined in a murine model. The extract acts, on the one hand, as macrophage activator and polyclonal B-lymphocyte stimulant. On the other hand, after repeated intraperitoneal or oral immunizations, the extract is immunogenic, inducing serum IgG

W. G. Bessler; M. Huber; W. Baier

1997-01-01

112

Antibacterial Activities of Crude Extract of Aloe barbadensis to Clinically Isolated Bacterial Pathogens  

Microsoft Academic Search

The antibacterial activity of Aloe barbadensis was tested on clinically isolated bacterial pathogens i.e. Enterococcus bovis, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa,\\u000a Morganella morganii, and Klebsiella pneumoniae causing infection in human being. Ethanolic and aqueous extracts were used for the antibacterial effect, which was measured\\u000a by the appearance of zone of inhibition. Relatively higher MIC concentrations

Ruchi Pandey; Avinash Mishra

2010-01-01

113

Bacterial model system for screening and determining optimal concentration of anti-caries natural extracts.  

PubMed

In general, an antimicrobial test for screening anti-caries natural extracts was performed by measuring the minimum bactericidal concentration (MBC) against the type strains of mutans streptococci. However, it is unclear if the antimicrobial efficiency of natural extracts on the type strains of mutans streptococci is the same on the clinical strains. In this study, we introduced a bacterial model system for the screening of anti-caries and determining the optimal concentration of them to develop oral hygiene products for Korean populations. PMID:21369996

Kim, Min Jung; Kim, Chun Sung; Park, Jae-Yoon; Park, Soon-Nang; Yoo, So Young; Lee, Sook-Young; Kook, Joong-Ki

2011-03-03

114

Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts  

PubMed Central

Here we describe a northern blot procedure that allows the detection of endogenous RNAs as small as ?14 nt in total RNA extracts from bacteria. RNAs that small and as part of total bacterial RNA extracts usually escape detection by northern blotting. The approach combines LNA probes 5?-digoxigenin-endlabeled for non-radioactive probe detection with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-mediated chemical crosslinking of RNAs to nylon membranes, and necessitates the use of native PAGE either with the TBE or MOPS buffer system.

Beckmann, Benedikt M.; Grunweller, Arnold; Weber, Michael H. W.; Hartmann, Roland K.

2010-01-01

115

Optimising Bacterial DNA Extraction from Faecal Samples: Comparison of Three Methods  

PubMed Central

Culture independent methods are used widely in diagnostic laboratories for infectious disease Isolation of genomic DNA from clinical samples is the first and important step in the procedure. Several procedures for extracting DNA from faecal samples have been described, including different mechanical cell disruptors. To our knowledge, the use of TissueLyser as a mechanical disruptor on faecal samples before DNA extraction has not been previously described. The purpose of the study was to implement a method for preparing faecal samples for optimal DNA extraction. Thus, three different procedures for extracting DNA from human faeces were compared. This was done either by using the mechanical disrupter by Mini BeadBeater 8, or the TissueLyser both followed by DNA purification using QIAamp DNA stool MiniKit, in comparison with DNA extractions using QIAamp DNA stool MiniKit without any prior mechanical disruption, according to manufacturer’s instructions. The obtained DNA from the three procedures was analysed by DGGE, and the number of bands was compared between each procedure. There was no significant difference between the numbers of bacterial bands obtained from DGGE when using a TissueLyser or Mini BeadBeater 8, so the two different mechanical cell disruptors can be used comparably when isolating bacterial DNA from faecal samples. The QIAamp DNA stool MiniKit alone resulted in a reduced number of bands compared to the two mechanical disruption methods.

Smith, Birgitte; Li, Nan; Andersen, Anders Schou; Slotved, Hans Christian; Krogfelt, Karen Angeliki

2011-01-01

116

In vitro antibacterial potential of Eugenia jambolana seed extracts against multidrug-resistant human bacterial pathogens.  

PubMed

The present study was carried out to evaluate the possible in vitro antibacterial potential of extracts of Eugenia jambolana seeds against multidrug-resistant human bacterial pathogens. Agar well diffusion and microbroth dilution assay methods were used for antibacterial susceptibility testing. Kill-kinetics study was done to know the rate and extent of bacterial killing. Phytochemical analysis and TLC-bioautography were performed by colour tests to characterize the putative compounds responsible for this antibacterial activity. Cytotoxic potential was evaluated on human erythrocytes by haemolytic assay method and acute oral toxicity study was done in mice. The plant extracts demonstrated varying degrees of strain specific antibacterial activity against all the test isolates. Further, ethyl acetate fraction obtained from fractionation of most active ethanol extract showed maximum antibacterial effect against all the test isolates. Phytochemical analysis and TLC-bioautography of ethyl acetate fraction revealed that phenolics were the major active phytoconstituents. Ethyl acetate fraction also demonstrated no haemolytic activity on human erythrocytes and no gross behavioural changes as well as toxic symptoms were observed in mice at recommended dosage level. The results provide justification for the use of E. jambolana in folk medicine to treat various infectious diseases and may contribute to the development of novel antimicrobial agents for the treatment of infections caused by these drug-resistant bacterial pathogens. PMID:22444436

Bag, Anwesa; Bhattacharyya, Subir Kumar; Pal, Nishith Kumar; Chattopadhyay, Rabi Ranjan

2012-03-22

117

16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran.  

PubMed

Nukadoko is a naturally fermented rice bran mash traditionally used for pickling vegetables in Japan; its refreshment and fermentation cycles sometimes continue for many years. Here, we investigated the structure and dynamics of the bacterial community in nukadoko by conducting pyrosequencing and quantitative polymerase chain reaction (PCR) analyses of 16S ribosomal RNA genes (rDNA). Of the 16 different samples studied, 13 showed Lactobacillus-dominated microbiota, suggesting that aged nukadoko samples tend to realize a niche, favorable Lactobacillus species. The lactic acid bacterial community of each of the 16 samples was classified into 3 types according to the presence or absence of 2 predominant species, Lactobacillus namurensis and Lactobacillus acetotolerans. The dynamics of the bacterial community during fermentation and the subsequent ripening process were examined using a laboratory model of nukadoko inoculated with an aged nukadoko sample (inoculated model). Lb. namurensis grew rapidly in the first 2 days, accompanied with a rapid decrease in pH and an increase in lactate levels, while Lb. acetotolerans grew with a longer doubling time and slow acidification during the 20 days after inoculation. On the other hand, spontaneous fermentation of the nukadoko model prepared from fresh rice bran without the nukadoko inoculation (inoculant-free model), showed the growth of some non-Lactobacillus species such as staphylococci and bacilli within the first 10 days; thereafter, Lb. namurensis was dominant, while Lb. acetotolerans was not detected during the 20-day experimental period. These results suggest that the naturally established Lactobacillus community in aged nukadoko is effectively involved in the biocontrol of the microbial community of nukadoko during the refreshment and fermentation cycles. PMID:21084126

Sakamoto, Naoshige; Tanaka, Shigemitsu; Sonomoto, Kenji; Nakayama, Jiro

2010-11-16

118

Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR  

PubMed Central

The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4°C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 × 105-fold, 1.5 × 105-fold, and 1.0 × 104-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

2005-01-01

119

Comparison of DNA extraction methods in analysis of salivary bacterial communities.  

PubMed

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used. PMID:23844068

Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

2013-07-03

120

Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis  

Microsoft Academic Search

Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then

Antonio Gelsomino; Anneke C Keijzer-Wolters; Giovanni Cacco; Jan Dirk van Elsas

1999-01-01

121

Phylogenetic Clustering of Soil Microbial Communities by 16S rRNA but Not 16S rRNA Genes  

PubMed Central

We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA.

Firestone, Mary K.

2012-01-01

122

Structure and dynamics of the bacterial communities in fermentation of the traditional Chinese post-fermented pu-erh tea revealed by 16S rRNA gene clone library.  

PubMed

Microbes are thought to have key roles in the development of the special properties of post-fermented pu-erh tea (pu-erh shucha), a well-known traditional Chinese tea; however, little is known about the bacteria during the fermentation. In this work, the structure and dynamics of the bacterial community involved in the production of pu-erh shucha were investigated using 16S rRNA gene clone libraries constructed from samples collected on days zero (LD-0), 5 (LD-5), 10 (LD-10), 15 (LD-15) and 20 (LD-20) of the fermentation. A total of 747 sequences with individual clone library containing 115-174 sequences and 4-20 unique operational taxonomic units (OTUs) were obtained. These OTUs were grouped into four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and further identified as members of 10 families, such as Alcaligenaceae, Bacillaceae, Enterobacteriaceae, etc. The dominant bacteria were Enterobacteriaceae in the raw material (LD-0) and in the initial stages of fermentation (LD-5 and LD-10), which changed to Bacillaceae at the last stages of fermentation (LD-15 and LD-20) at a temperature of 40-60 °C. It is interesting that the dominant OTUs in libraries LD-15 and LD-20 were very closely related to Bacillus coagulans, which is a safe thermoduric probiotic. Together the bacterial diversity and dynamics during a fermentation of pu-erh shucha were demonstrated, and a worthy clue for artificial inoculation of B. coagulans to improve the health benefits of pu-erh shucha or produce probiotic pu-erh tea were provided. PMID:23591759

Zhao, Ming; Xiao, Wei; Ma, Yan; Sun, Tingting; Yuan, Wenxia; Tang, Na; Zhang, Donglian; Wang, Yongxia; Li, Yali; Zhou, Hongjie; Cui, Xiaolong

2013-04-17

123

Phylogenetic Analysis of Rumen Bacteria by Comparative Sequence Analysis of Cloned 16S rRNA Genes ß  

Microsoft Academic Search

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage\\/corn silage\\/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In

Marc F Whitford; Robert J Forster; Cheryl E Beard; Jianhua Gong; Ronald M Teather

1998-01-01

124

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad  

Microsoft Academic Search

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library

Elena Barbieri; Luana Bertini; Ismaela Rossi; Paola Ceccaroli; Roberta Saltarelli; Chiara Guidi; Alessandra Zambonelli; Vilberto Stocchi

2005-01-01

125

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.  

PubMed

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods. PMID:16621089

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-04-18

126

Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing  

SciTech Connect

The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host plants (5,27,40). Tatineni and colleagues discovered that the HLB bacteria were unevenly distributed in phloem of bark tissue, vascular tissue of the leaf midrib, roots, and different floral and fruit parts (43). Unsuccessful attempts in culturing the pathogen are notably hampering efforts to understand its biology and pathogenesis mechanism. Using a modified Koch's Postulates approach, Jagoueix and colleagues were able to re-infect periwinkle plants from a mixed microbial community harvested from HLB diseased plants (25). Emergence of the disease in otherwise healthy plants led to the conclusion that HLB was associated with Candidatus Liberibacter sp. based on its 16S rDNA sequence (18,25). Currently, three species of the pathogen are recognized from trees with HLB disease based on 16S rDNA sequence: Ca. Liberibacter asiaticus (Las), Ca. Liberibacter africanus (Laf), and Ca. Liberibacter americanus (Lam); Las is the most prevalent species among HLB diseased trees (5,12,18,25,44). Las is naturally transmitted to citrus by the psyllid, Diaphorina citri Kuwayama, and can be artificially transmitted by grafting from citrus to citrus and dodder (Cuscuta campestris) to periwinkle (Catharanthus roseus) or tobacco (Nicotiana tabacum Xanthi) (5). Based on current research regarding the associations of Liberibacter in planta there is not enough evidence to implicate Liberibacter as the definitive causal agent of HLB disease due to its resistance to cultivation in vitro. It is possible that HLB disease may be the result of complex etiology where Liberibacter interacts with other endophytic bacteria. However, there is not enough evidence regarding its association(s) in planta to make this conclusion, nor is it known whether associated microbial communities play a role in expression of pathogenic traits. The main objective of the study was to test the hypothesis that other bacteria besides Ca. Liberibacter spp. are associated with citrus greening disease. The differences between the relative abundance, species richness and phylogenetic diversity of the microbial communitie

Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

2009-03-01

127

Potential applications for Annona squamosa leaf extract in the treatment and prevention of foodborne bacterial disease.  

PubMed

Foodborne disease is a major public health problem. The present study examined Annona squamosa leaves, which are traditionally used to treat diarrhea and other infections, for their potential to be used in modern food safety or medicine. Active constituents were partially purified by ethanol extraction and column chromatography. MICs of the extract were 62.5 to 125 microg/mL against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and 250 microg/mL against Campylobacter jejuni. In time-kill assays, 500 microg/mL of the extract reduced colony forming unit numbers of C. jejuni almost 10 000-fold within 12 hours. Similar decreases were seen against B. cereus, but over a longer time-frame. LC-MS analysis indicated the presence of reticuline and oxophoebine. Assessment of stability by MIC assay showed activity was heat-labile, with loss of activity greatest following high temperature treatments. Activity was relatively stable at refrigeration temperature. These results indicate A. squamosa has broad-spectrum but heat-labile activity against foodborne bacterial pathogens, and bactericidal activity against B. cereus and C. jejuni. This bactericidal activity is not sufficiently rapid for A. squamosa to be used as a food sanitizer, but the extract could potentially be developed as an additive for refrigerated foods, or a modern treatment for foodborne illness. PMID:23678817

Dholvitayakhun, Achara; Trachoo, Nathanon; Sakee, Uthai; Cushnie, T P Tim

2013-03-01

128

Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. [Humans  

SciTech Connect

The genotoxicities of organic extracts of airborne particles have been studied extensively in the Salmonella/mammalian microsome (Ames) test, but in few other bioassays. In these studies, we tested benzene-acetone extracts of particulate pollutants collected in Lexington, Kentucky, for capacity to induce increases in sister chromatid exchanges (SCE) in human lumphocytes and V79 cells, as well as in the Ames assay. Extracts induced linear dose-related increases in SCE in human lumphocytes and in bacterial revertants.However, variable responses were observed in SCE assays in V79 cells with and without activation by rat liver S9 or feeder layers of irradiated Syrian hamster fetal cells. We conclude that the SCE assay in human lumphocytes may be a useful indicator of the potential risks to humans of airborne particulate pollutants, as it utilizes human cells recently taken from the host, is rapid and economical, and requires small quantities of test materials. However, thorough studies of the quantitative relationships between SCE induction and mutagenicity in human cells are needed.

Lockard, J.M. (Thomas Hunt Morgan School of Biological Sciences, Lexington, KY); Viau, C.J.; Lee-Stephens, C.; Caldwell, J.C.; Wojciechowski, J.P.; Enoch, H.G.; Sabharwal, P.S.

1981-01-01

129

Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract.  

PubMed

Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L. albus and L. luteus had been grown [Santana, F. M. et al. J. Ind. Microbiol. 1996, 17, 110-115]. In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C. Growth in L. albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)). This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids. Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L. albus extract did not vary significantly. PMID:11929291

Santana, Filomena M C; Pinto, Teresa; Fialho, Arsénio M; Sá-Correia, Isabel; Empis, José M A

2002-04-10

130

Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment  

Microsoft Academic Search

The assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designated to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results

SUSAN M. BARNS; SHANNON L. TAKALA; CHERYL R. KUSKE

1999-01-01

131

Archaeal and bacterial communities in geochemically diverse hot springs of Yellowstone National Park, USA  

Microsoft Academic Search

Microbiological and geochemical surveys were conducted at three hot springs (Obsidian Pool, Sylvan Spring, and 'Bison Pool') in Yellowstone National Park (Wyoming, USA). Microbial community structure was investi- gated by polymerase chain reaction (PCR) amplification of 16S rRNA gene sequences from DNA extracted from sediments of each hot spring, followed by molecular cloning. Both bacterial and archaeal DNA was retrieved

D. R. MEYER-DOMBARD; E. L. SHOCK; J. P. AMEND

2005-01-01

132

Antibacterial efficacy of the seed extracts of Melia azedarach against some hospital isolated human pathogenic bacterial strains  

PubMed Central

Objective To investigate the antibacterial potential of the polar and non-polar extracts of the seeds of Melia azedarach (M. azedarach) L. (Meliaceae) against eighteen hospital isolated human pathogenic bacterial strains. Methods Petrol, benzene, ethyl acetate, methanol, and aqueous extracts at five different concentrations (1, 2, 5, 10 and 15 mg/mL) were evaluated. Disk diffusion method was followed to evaluate the antibacterial efficacy. Results All extracts of the seeds demonstrated significant antibacterial activity against tested pathogens. Among all extracts, ethyl acetate extract revealed the highest inhibition comparatively. The present study also favored the traditional uses reported earlier. Conclusions Results of this study strongly confirm that the seed extracts of M. azedarach could be effective antibiotics, both in controlling gram-positive and gram-negative human pathogenic infections.

Khan, Abdul Viqar; Ahmed, Qamar Uddin; Mir, M Ramzan; Shukla, Indu; Khan, Athar Ali

2011-01-01

133

Bacterial diversity in the intestinal tract of the fungus- cultivating termite Macrotermes michaelseni (Sjöstedt)  

Microsoft Academic Search

Microorganisms in the intestinal tracts of termites play a crucial role in the nutritional physiology of termites. The bacterial diversity in the fungus-cultivating Macrotermes michaelseni was examined using both molecular and culture dependent methods. Total DNA was extracted from the gut of the termite and 16S rRNA genes were amplified using bacterial specific primers. Representatives from forty-one (41) RFLP patterns

Lucy Mwende Mackenzie; Anne Thairu Muigai; Ellie Onyango Osir; Wilber Lwande; Martin Keller; Gerardo Toledo; Hamadi Iddi Boga

2007-01-01

134

Using Bacterial Extract along with Differential Gene Expression in Acropora millepora Larvae to Decouple the Processes of Attachment and Metamorphosis  

PubMed Central

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0–2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05) in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05) following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that utilized cues, such as crustose coralline algae, biofilms or with GLW-amide neuropeptides that stimulate the entire onset of larval metamorphosis and attachment.

Siboni, Nachshon; Abrego, David; Seneca, Francois; Motti, Cherie A.; Andreakis, Nikos; Tebben, Jan; Blackall, Linda L.; Harder, Tilmann

2012-01-01

135

Absence of mutagenic effects of a particular Symphytum officinale L. liquid extract in the bacterial reverse mutation assay.  

PubMed

Comfrey (Symphytum officinale L.) root is traditionally used for the topical treatment of contusions, strains and sprains. Besides allantoin and rosmarinic acid, which are discussed as pharmacologically active principles, the drug contains pyrrolizidine alkaloids (PAs) known for their hepatotoxic, carcinogenic and mutagenic properties. The topical herbal medicinal products Kytta-Salbe f and Kytta-Plasma f contain a PA-free liquid extract from comfrey root as active substance. The aim of this study was to demonstrate the absence of genotoxic effects of this special extract in the bacterial reverse mutation assay (Ames test). Briefly, comfrey root liquid extract was investigated for its ability to induce gene mutations in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation using the mammalian microsomal fraction S9 mix. Reference mutagens were used to check the validity of the experiments. Comfrey root fluid extract showed no biologically relevant increases in revertant colony numbers of any of the five tester strains, neither in the presence nor in the absence of metabolic activation. In conclusion, the comfrey root fluid extract contained in Kytta-Salbe f and Kytta-Plasma f was not mutagenic in the bacterial reverse mutation assay. PMID:19827020

Benedek, Birgit; Ziegler, Andreas; Ottersbach, Peter

2010-03-01

136

Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis  

Microsoft Academic Search

The bacterial diversity and population dynamics in the rhizosphere of two maize cultivars (Nitroflint and Nitrodent) grown in tropical soils was studied, by traditional cultivation techniques and 16S rRNA gene-based molecular analysis of DNA directly extracted from soil and rhizosphere samples. Rhizosphere and soil samples were taken at three different plant growth stages. Total aerobic bacterial counts were determined. Fingerprints

N. C. M. Gomes; H. Heuer; J. Schönfeld; R. Costa; L. Mendonça-Hagler; K. Smalla

2001-01-01

137

Intragenomic and intraspecific heterogeneity of the 16S rRNA gene in seven bacterial species from the respiratory tract of cystic fibrosis patients assessed by PCR-Temporal Temperature Gel Electrophoresis.  

PubMed

16S rRNA gene-based cultivation-independent methods are increasingly used to study the diversity of microbiota during health and disease. One bias of these methods is the variability of 16S rRNA gene that may exist among strains of a same species (intraspecific heterogeneity) or between rrs copies in a genome (intragenomic heterogeneity). We evaluated the level of intraspecific and intragenomic 16S rDNA variability in seven species frequently encountered in respiratory tract samples in cystic fibrosis (CF). A total of 179 strains were subjected to V3 region 16S rDNA PCR-TTGE. Using this easy-to-perform and rapid method, different levels of V3 region rrs heterogeneity were demonstrated. No intraspecific and intragenomic rrs heterogeneity was demonstrated for Moraxella catarrhalis (n=16), Pseudomonas aeruginosa (n=31) and Streptococcus pneumoniae (n=14) showing a single PCR-TTGE band characteristic of the species. Low level of intraspecific heterogeneity was observed for Staphylococcus aureus (n=30), Stenotrophomonas maltophilia (n=29) and Achromobacter xylosoxidans (n=28), and 17%, 38% and 96% of these strains showed intragenomic heterogeneity (two to four different rrs copies), respectively. Haemophilus influenzae (n=31) displayed the higher level of intraspecific variability with 23 different PCR-TTGE patterns and 61% of the strains showed intragenomic rrs heterogeneity (two to four different rrs copies). Although only one hypervariable region of the 16S rRNA gene was explored, intraspecific and intragenomic rrs heterogeneity was frequently observed in this study and should be taken into consideration for a better interpretation of 16S rRNA gene-based diversity profiles in denaturing gels and to avoid any overestimation of the respiratory microbiota diversity in CF. PMID:21621347

Michon, A-L; Jumas-Bilak, E; Imbert, A; Aleyrangues, L; Counil, F; Chiron, R; Marchandin, H

2011-05-31

138

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA  

Microsoft Academic Search

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA

Hikaru Suenaga; Rui Liu; Yuko Shiramasa; Takahiro Kanagawa

2005-01-01

139

Life History Implications of rRNA Gene Copy Number in Escherichia coli  

Microsoft Academic Search

The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of Escherichia coli in which one or two of the seven rRNA operons (rrnA and\\/or rrnB) were deleted. The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures. In batch cultures, the

Bradley S. Stevenson; Thomas M. Schmidt

2004-01-01

140

Selective Enhancement of Systemic Th1 Immunity in Immunologically Immature Rats with an Orally Administered Bacterial Extract  

PubMed Central

Infant rats primed during the first week of life with soluble antigen displayed adult-equivalent levels of T-helper 2 (Th2)-dependent immunological memory development as revealed by production of secondary immunoglobulin G1 (IgG1) antibody responses to subsequent challenge, but in contrast to adults failed to prime for Th1-dependent IgG2b responses. We demonstrate that this Th2 bias in immune function can be redressed by oral administration to neonates of a bacterial extract (Broncho-Vaxom OM-85) comprising lyophilized fractions of several common respiratory tract bacterial pathogens. Animals given OM-85 displayed a selective upregulation in primary and secondary IgG2b responses, accompanied by increased gamma interferon and decreased interleukin-4 production (both antigen specific and polyclonal), and increased capacity for development of Th1-dependent delayed hypersensitivity to the challenge antigen. We hypothesize that the bacterial extract functions via enhancement of the process of postnatal maturation of Th1 function, which is normally driven by stimuli from the gastrointestinal commensal microflora.

Bowman, L. M.; Holt, P. G.

2001-01-01

141

Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration  

Microsoft Academic Search

The aim of this study was to screen and evaluate the antimicrobial activity of indigenous Jordanian plant extracts, dissolved in dimethylsulfoxide, using the rapid XTT assay and viable count methods. XTT rapid assay was used for the initial screening of antimicrobial activity for the plant extracts. Antimicrobial activity of potentially active plant extracts was further assessed using the “viable plate

Amal G. Al-Bakri; Fatma U. Afifi

2007-01-01

142

Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria  

PubMed Central

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (?, ?, and ? subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR–single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.

Schwieger, Frank; Tebbe, Christoph C.

2000-01-01

143

The oral administration of bacterial extracts prevents asthma via the recruitment of regulatory T cells to the airways.  

PubMed

The prevalence of asthma has steadily increased during the last decade, probably as the result of changes in the environment, including reduced microbial exposure during infancy. Accordingly, experimental studies have shown that deliberate infections with live pathogens prevent the development of allergic airway diseases in mice. Bacterial extracts are currently used in children suffering from repeated upper respiratory tract infections. In the present study, we have investigated whether bacterial extracts, commercially available as Broncho-Vaxom (BV), could prevent allergic airway disease in mice. Oral treatment with BV suppressed airway inflammation through interleukin-10 (IL-10)-dependent and MyD88 (myeloid differentiation primary response gene (88))-dependent mechanisms and induced the conversion of FoxP3 (forkhead box P3)(-) T cells into FoxP3(+) regulatory T cells. Furthermore, CD4(+) T cells purified from the trachea of BV-treated mice conferred protection against airway inflammation when adoptively transferred into sensitized mice. Therefore, treatment with BV could possibly be a safe and efficient strategy to prevent the development of allergic diseases in children. PMID:20811345

Navarro, S; Cossalter, G; Chiavaroli, C; Kanda, A; Fleury, S; Lazzari, A; Cazareth, J; Sparwasser, T; Dombrowicz, D; Glaichenhaus, N; Julia, V

2010-09-01

144

Use of the MicroSeq 500 16S rRNA Gene-Based Sequencing for Identification of Bacterial Isolates That Commercial Automated Systems Failed To Identify Correctly  

PubMed Central

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux). However, more and more frequently, microbiologists must isolate “difficult” strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 “difficult” clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems—VITEK 2 and Phoenix—while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.

Fontana, Carla; Favaro, Marco; Pelliccioni, Marco; Pistoia, Enrico Salvatore; Favalli, Cartesio

2005-01-01

145

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces.  

PubMed

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers. PMID:18162191

Nechvatal, Jordan M; Ram, Jeffrey L; Basson, Marc D; Namprachan, Phanramphoei; Niec, Stephanie R; Badsha, Kawsar Z; Matherly, Larry H; Majumdar, Adhip P N; Kato, Ikuko

2007-11-21

146

High aromatic ring-cleavage diversity in birch rhizosphere: PAH treatment-specific changes of I.E.3 group extradiol dioxygenases and 16S rRNA bacterial communities in soil  

Microsoft Academic Search

Genes encoding key enzymes of catabolic pathways can be targeted by DNA fingerprinting to explore genetic degradation potential in pristine and polluted soils. We performed a greenhouse microcosm experiment to elucidate structural and functional bacterial diversity in polyaromatic hydrocarbon (PAH)-polluted soil and to test the suitability of birch (Betula pendula) for remediation. Degradation of PAHs was analysed by high-performance liquid

Timo P Sipilä; Anna-Kaisa Keskinen; Marja-Leena Åkerman; Carola Fortelius; Kielo Haahtela; Kim Yrjälä

2008-01-01

147

Effect of Solvent and Extraction Methods on the Bacterial Mutagenicity of Sidestream Cigarette Smoke.  

National Technical Information Service (NTIS)

The mutagenic activity of sidestream cigarette smoke particles was estimated by testing sidestream cigarette smoke particles which had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultr...

R. S. Morin J. J. Tulis L. D. Claxton

1987-01-01

148

Plant extracts for the control of bacterial growth: efficacy, stability and safety issues for food application.  

PubMed

The microbial safety of foods continues to be a major concern to consumers, regulatory agencies and food industries throughout the world. Many food preservation strategies have been used traditionally for the control of microbial spoilage in foods but the contamination of food and spoilage by microorganisms is a problem yet to be controlled adequately. Although synthetic antimicrobials are approved in many countries, the recent trend has been for use of natural preservatives, which necessitates the exploration of alternative sources of safe, effective and acceptable natural preservatives. Plants contain innumerable constituents and are valuable sources of new and biologically active molecules possessing antimicrobial properties. Plants extracts either as standardized extracts or as a source of pure compounds provide unlimited opportunities for control of microbial growth owing to their chemical diversity. Many plant extracts possess antimicrobial activity against a range of bacteria, yeast and molds, but the variations in quality and quantity of their bioactive constituents is the major detriments in their food use. Further, phytochemicals added to foods may be lost by various processing techniques. Several plant extracts or purified compounds intended for food use have been consumed by humans for thousands of years, but typical toxicological information is not available for them. Although international guidelines exist for the safety evaluation of food additives, owing to problems in standardization of plant extracts, typical toxicological values have not been assigned to them. Development of cost effective isolation procedures that yield standardized extracts as well as safety and toxicology evaluation of these antimicrobials requires a deeper investigation. PMID:22459761

Negi, Pradeep Singh

2012-03-11

149

Estimation of the Abundance of an Uncultured Soil Bacterial Strain by a Competitive Quantitative PCR Method  

Microsoft Academic Search

Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample

SOO-YOUN LEE; JOHN BOLLINGER; DAVID BEZDICEK; ANDANDREW OGRAM

1996-01-01

150

Automated extraction of typing information for bacterial pathogens from whole genome sequence data: Neisseria meningitidis as an exemplar.  

PubMed

Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely phenotypes of aetiological agents, enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that many microbiological typing methods have addressed over the last 100 years or so; indeed, providing large volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites have generated an infrastructure that can exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST website to extract clinically useful, strain-characterisation information that can be provided to physicians and public health professionals in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to instigate public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data obtained in the absence of WGS, providing a flexible infrastructure for WGS-based clinical microbiology. PMID:23369391

Jolley, K A; Maiden, M C

2013-01-24

151

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.  

PubMed

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria. PMID:16684100

Nakimbugwe, Dorothy; Masschalck, Barbara; Deckers, Daphne; Callewaert, Lien; Aertsen, Abram; Michiels, Chris W

2006-06-01

152

Inhibition of bacterial quorum sensing-regulated behaviors by Tremella fuciformis extract.  

PubMed

Quorum sensing (QS), or the control of gene expression in response to cell density, is used by both gram-negative and gram-positive bacteria to regulate a variety of physiological functions. Increasing evidence implies that certain eukaryotes produce QS-inhibitory compounds. In this work, we tested Tremella fuciformis for their ability to inhibit QS-regulated behaviors. T. fuciformis fruiting bodies were dried and extracted using 75% (v/v) aqueous methanol. The crude extract was redissolved in appropriate concentrations of dimethyl sulfoxide (DMSO), sterilized by filtration through a 0.45-microm membrane filter and added to Chromobacterium violaceum CV026 cultures, which was used to monitor QS inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. The results have revealed that the DMSO-soluble compounds extracted from T. fuciformis could inhibit violacein production, a QS-regulated behavior in C. violaceum. The results suggest an attractive tool to control and handle detrimental infections caused by human, animal, and plant pathogens. Further studies are required to isolate specific substances from T. fuciformis extract acting as QS inhibitors. PMID:18661179

Zhu, H; Sun, S J

2008-07-26

153

Interaction of plant extracts of Ungernia victoris, Rhodiola rosea , and Polyscias filicifolia with a bacterial cell  

Microsoft Academic Search

Components of three investigated plant extracts from the biomass of cultivated cells of Ungernia victoris, Rhodiola rosea, and Polyscias filicifolia interact with porin proteins OmpC and OmpF and reduce their receptor activity towards OmpC- and OmpF-dependent bacteriophages.\\u000a The influx of these components into cells optimizes the synthesis of a lipopolysaccharide (LPS) complex and contributes to\\u000a the enhancement of receptor activity

T. P. Pererva; A. Yu. Miryuta; L. P. Mozhylevskaya; V. A. Kunakh

2010-01-01

154

Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages  

Microsoft Academic Search

In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from

LUCA COCOLIN; MARISA MANZANO; CARLO CANTONI; GIUSEPPE COMI

2001-01-01

155

An in vitro study of polymorphonuclear leucocyte-mediated injury to human gingival keratinocytes by periodontopathic bacterial extracts.  

PubMed

Human gingival keratinocytes were cultured and, after the first passage, subjected to cell detachment assays with polymorphonuclear leucocytes (PMNs) and/or sonic extracts from Actinobacillus actinomycetemcomitans Y4, and Eikenella corrodens 1073. The effector-to-target cell ratio was 30:1. Bacterial extracts alone caused no disruption of keratinocyte monolayers. PMNs alone also caused only minimal detachment after 14 h incubation. Adding A. actinomycetemcomitans to the PMN-keratinocyte co-cultures at the concentration of 100 micrograms/ml caused dramatic cell detachment. The effect of A. actinomycetemcomitans was heat labile and not inhibited by polymyxin B. Cell detachment was inhibited by alpha 1-antitrypsin, whereas catalase and superoxide dismutase could not prevent it. No lysis of keratinocytes was observed after incubation, as judged by 51Cr release. E. corrodens had little effect even at the concentration of 1000 micrograms/ml. H2O2 and partially purified PMN elastase also caused detachment of keratinocytes. These data indicate that PMNs can cause non-lytic detachment of keratinocytes when interacting with certain bacteria. PMID:1471949

Sugiyama, E; Baehni, P; Cimasoni, G

1992-12-01

156

Evaluation of novel carbon nano-tube particles in the bacterial and viral DNA and RNA extraction from the clinical samples  

Microsoft Academic Search

Molecular techniques have become the most im- portant methods of detecting bacterial and viral pathogens. However, current genomic extraction methods are currently limited in term of automation. In this study, carbon nano-tube was used as the vector to trap DNA and RNA molecules. The capability of carbon nano-tube to trap DNA and RNA was evaluated using samples (TB and HBV

Nguyen KC; Vo DXA; Hoang HN; Ho LTT; Pham HV

2010-01-01

157

Phytochemical and anti-bacterial activity of epidermal glands extract of Christella parasitica (L.) H. Lev.  

PubMed Central

Objective To study the morphology, biochemistry and bioactivity of the epidermal glands of the glandular morphotype of Christella parasitica (C. parasitica) (L.) H. Lev. Methods Morphological studies on epidermal glands were carried out by using light microscope and scanning electron microscope. To prepare the extract, the shade-dried fronds of glandular morphotype were soaked in acetone. For antibacterial studies paper disc method was followed by using various pathogenic bacteria. Results Detailed micromorphological, phytochemical and bioactivity studies on a medicinal fern C. parasitica (L.) H. Lev. showed its intraspecific variation in antibacterial activity. The presence or absence of the epidermal glands was the key factor for antibacterial activity in the morphovariants of this species. The epidermal glands were orange-coloured, stalked and elongated ones of about 84.2 µm × 45 µm, and distributed on the undersurface of costa, costules and veins in croziers, young and mature leaves. Frequency of glands varied from 15/cm on costa in mature leaves to 140/cm on costules in croziers. The acetone extract of the glands showed antibacterial activities and also toxic effect against mosquito larvae and tadpoles of frog. Preliminary phytochemical analysis and HPLC studies of the gland extract showed the presence of various kinds of terpenoids, alkaloids, tannins, saponins and flavonoids in it. Conclusions The present study shows that epidermal glands of the glandular morphotype of C. parasitica (L.) H. Lev. have several bioactive compounds and such rare morphovariant should be conserved in nature. The next step is to isolate the pure compounds and to screen the bioactivity of individual compounds of the epidermal glands.

Paul, Raj K; Irudayaraj, V; Johnson, M; Patric, Raja D

2011-01-01

158

Folding of 16S rRNA in a Signal-Producing Structure for the Detection of Bacteria.  

PubMed

Sixty-four DNA strands hybridize to 16S rRNA to form 32 deoxyribozyme catalytic cores that produce a fluorescent signal. The approach allows detection of 0.6?pM 16S rRNA, or about 3×10(4) bacterial cells in a PCR-free format. PMID:24038733

Gerasimova, Yulia V; Kolpashchikov, Dmitry M

2013-09-03

159

Protective effect of Polygonum orientale L. extracts against Clavibater michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato.  

PubMed

The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L(27)3(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80°C, solid to liquid ratio 1?10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease. PMID:23861908

Cai, Jin; Xie, Shulian; Feng, Jia; Wang, Feipeng; Xu, Qiufeng

2013-07-04

160

Protective Effect of Polygonum orientale L. Extracts against Clavibater michiganense subsp. sepedonicum, the Causal Agent of Bacterial Ring Rot of Potato  

PubMed Central

The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L273(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80°C, solid to liquid ratio 1?10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease.

Cai, Jin; Xie, Shulian; Feng, Jia; Wang, Feipeng; Xu, Qiufeng

2013-01-01

161

rRNA Specific Oligonucleotides.  

National Technical Information Service (NTIS)

The invention relates to a method of inhibiting protein synthesis comprising contacting 28S rRNA of a protein synthesizing system with a protein synthesis inhibitory amount of an oligonucleotide that hybridizes to the alpha-sarcin recognition domain loop ...

E. J. Ackerman

1989-01-01

162

Methods for extracting biochemical information from bacterial Raman spectra: an explorative study on Cupriavidus metallidurans.  

PubMed

In this study, we explore Raman spectra of bacteria for their biochemical information. Therefore, a database of biomolecules was used and several approaches were applied such as the study of difference spectra, the calculation dot products, the usage of coefficients obtained from an EMSC procedure and the application of 2D correlation spectroscopy. These methods were applied on a dataset containing Raman spectra of Cupriavidus metallidurans LMG 1195 in five stages of its growth, aiming to extract information about the evolution of cell components during growth. EMSC coefficients seemed to be most promising for tracking metabolic products and the results were often confirmed by difference spectra or by 2D correlation spectroscopy. PMID:17386670

De Gelder, Joke; De Gussem, Kris; Vandenabeele, Peter; De Vos, Paul; Moens, Luc

2007-01-12

163

Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract  

PubMed Central

A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.

Jeuken, Lars J.C.; Connell, Simon D.; Nurnabi, Mohammed; O'Reilly, John; Henderson, Peter J.F.; Evans, Stephen D.; Bushby, Richard J.

2013-01-01

164

Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste.  

PubMed

Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments. PMID:19440973

Maciel, B M; Santos, A C F; Dias, J C T; Vidal, R O; Dias, R J C; Gross, E; Cascardo, J C M; Rezende, R P

2009-03-31

165

The majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides  

PubMed Central

We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacterially derived components when tested in in vitro monocyte/macrophage activation systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the majority (85–98%) of the activity within extracts from eight immune enhancing botanicals was eradicated by treatment with agents (lipoprotein lipase and polymyxin B) known to target these two bacterial components. Alfalfa sprouts exhibited the highest activity of those botanicals tested but the appearance of this activity during the germination of surface sterilized seeds was abolished by the presence of antibiotics. These studies indicate that the majority of the in vitro macrophage activating properties in extracts from these botanicals can be attributed to the presence of lipoproteins and lipopolysaccharides derived from bacteria and that bacterial endophytes may be a significant source of these components.

Pugh, Nirmal D.; Tamta, Hemlata; Balachandran, Premalatha; Wu, Xiangmei; Howell, J'Lynn; Dayan, Franck E.; Pasco, David S.

2008-01-01

166

rRNA Operon Copy Number Reflects Ecological Strategies of Bacteria  

Microsoft Academic Search

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond

JOEL A. KLAPPENBACH; JOHN M. DUNBAR; THOMAS M. SCHMIDT

2000-01-01

167

A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists  

PubMed Central

Background Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Principal Findings Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. Conclusions The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.

Marron, Alan O.; Akam, Michael; Walker, Giselle

2013-01-01

168

Detection of rifampin- and ciprofloxacin-resistant Mycobacterium tuberculosis by using species-specific assays for precursor rRNA.  

PubMed Central

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well.

Cangelosi, G A; Brabant, W H; Britschgi, T B; Wallis, C K

1996-01-01

169

Effect of Dietary Supplementation with a Saccharomyces cerevisiae Mannan Oligosaccharide on the Bacterial Community Structure of Broiler Cecal Contents?†  

PubMed Central

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure.

Corrigan, A.; Horgan, K.; Clipson, N.; Murphy, R. A.

2011-01-01

170

Effect of dietary supplementation with a Saccharomyces cerevisiae mannan oligosaccharide on the bacterial community structure of broiler cecal contents.  

PubMed

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure. PMID:21803917

Corrigan, A; Horgan, K; Clipson, N; Murphy, R A

2011-07-29

171

Succession of Microbial Communities during Hot Composting as Detected by PCR-Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes  

PubMed Central

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of ?-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

Peters, Sabine; Koschinsky, Stefanie; Schwieger, Frank; Tebbe, Christoph C.

2000-01-01

172

Target-Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens ? †  

PubMed Central

We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay.

Patel, Mayank; Gonzalez, Rodrigo; Halford, Colin; Lewinski, Michael A.; Landaw, Elliot M.; Churchill, Bernard M.; Haake, David A.

2011-01-01

173

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells  

Microsoft Academic Search

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the

Fekadu Kassie; Wolfram Parzefall; Stephen Musk; Ian Johnson; Günther Lamprecht; Gerhard Sontag; Siegfried Knasmüller

1996-01-01

174

The use of leucine incorporation to determine the toxicity of phenols to bacterial communities extracted from soil  

Microsoft Academic Search

The toxicity of different phenols to the soil bacterial community was studied in the laboratory using the leucine incorporation technique. The effects of environmental factors such as pH, temperature and binding strength to soil particles were also assessed in order to deduce confounding effects due to the chemical and physical conditions in the soil from which the bacterial community was

Louise Aldén Demoling; Erland Bååth

2008-01-01

175

Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 59Nuclease Assays  

Microsoft Academic Search

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5*-

MARCELINO T. SUZUKI; LANCE T. TAYLOR; EDWARD F. DELONG

2000-01-01

176

Changes of the bacterial assemblages throughout an urban drinking water distribution system.  

PubMed

We analyzed the bacterial 16S rRNA gene diversity throughout the major components of the drinking water distribution system of a ca. 52,000-inhabitants city (Trikala City, Greece) in order to describe the changes of the bacterial assemblages and to detect possible bacterial pathogens which are not included in the standard monitoring process. Bacterial DAPI counts and DNA extraction was performed in the water pumping wells, the water treatment tank and tap water from households. Approximately 920 bp of the bacterial 16S rDNA were PCR-amplified, cloned, and sequenced for a total of 191 clones, which belonged to 112 unique phylotypes. The water of the pumping wells harbored a typical subsurface bacterial assemblage, with no human pathogens, dominated by beta-Proteobacteria. Cell abundance in the water treatment tank decreased significantly, close to detection limit, but bacterial diversity remained high. However, the dominance of beta-Proteobacteria decreased considerably, indicating the sensitivity of this group to drinking water disinfection treatment. Tap water from the households hosted a much less diverse, low-cell bacterial assemblage, dominated by Mycobacterium-like phylotypes, related to biofilm bacterial communities. PMID:19404754

Kormas, Konstantinos Ar; Neofitou, Christos; Pachiadaki, Maria; Koufostathi, Eulalia

2009-04-30

177

Evaluation of extracts of Anthocleista djalonensis, Nauclea latifolia and Uvaria afzalii for activity against bacterial isolates from cases of non-gonococcal urethritis.  

PubMed

Whole root preparations of three Nigerian medicinal plants, Anthocleista djalonensis, Nauclea latifolia and Uvaria afzalii, used traditionally in combination treatment of sexually transmitted diseases (STD), were extracted by maceration in ethanol, cold and hot water, respectively. The extracts were tested, by agar diffusion and macrobroth dilution methods, for activity against five strains of Staphylococcus aureus and two of Escherichia coli isolated from cases of STD and or urethritis. Four typed bacterial strains, S aureus ATCC 12600, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa ATCC 10145 and Escherichia coli ATCC 117755 were included as reference organisms. Ethanolic and cold-water extracts of Anthocliesta djalonensis exhibited activity against 9 and 7, respectively, of the 11 test organisms. They were bacteriostatic at minimum inhibitory concentrations (MIC) to the Gram positive strains but bactericidal to the Gram negative strains. Similar crude extracts of Uvaria afzalii showed bactericidal activity restricted to Gram positive (Staphylococcus aureus and Bacillus subtilis) strains. Nauclea latifolia extracts were bacteriostatic to both Gram positive and Gram negative strains. No test strain was susceptible to the hot water extracts of Nauclea latifolia but five and seven strains, were respectively susceptible to similar extracts of Anthocliesta djalonensis and Uvaria afzalii. Of the seven column chromatographic fractions of the ethanolic extract of Uvaria afzalii, F(ua-1) exhibited a bactericidal activity restricted to the Gram negative Escherichia coli strains, which were not susceptible to the crude extract. Fractions, F(ua-2), F(ua-3) and F(ua-4), like the crude extract, were bactericidal against the Gram positive strains only. Thus, partial purification seems to broaden the spectrum of activity and generally improve the potency of Uvaria afzalii. These results apparently justify the use of the three plants in treatment of STD. PMID:15099860

Okoli, A S; Iroegbu, C U

2004-05-01

178

Towards complete biodiversity assessment: an evaluation of the subterranean bacterial communities in the Oklo region of the sole surviving natural nuclear reactor  

Microsoft Academic Search

Groundwater bacterial rRNA sequences extracted from the natural nuclear reactor region of Gabon are used to demonstrate the application of phylogenetic methods to biodiversity assessment. Clones were provisionally placed in `genera' using either the genus of the closest named EMBL entry, or by grouping clones at least 97.5% identical. The community is small, with 24 putative genera under the `closest-match'

R. H. Crozier; P.-M. Agapow; K. Pedersen

1999-01-01

179

A Mucoadhesive Polymer Extracted from Tamarind Seed Improves the Intraocular Penetration and Efficacy of Rufloxacin in Topical Treatment of Experimental Bacterial Keratitis  

PubMed Central

Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization. In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental Pseudomonas aeruginosa and Staphylococcus aureus keratitis in rabbits. The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes. Rufloxacin delivered by the polysaccharide reduced P. aeruginosa and S. aureus in the cornea at a higher rate than that obtained by rufloxacin alone. In particular, use of the polysaccharide allowed a substantial reduction of S. aureus in the cornea to be achieved even when the time interval between drug administrations was extended. These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs. The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis.

Ghelardi, Emilia; Tavanti, Arianna; Davini, Paola; Celandroni, Francesco; Salvetti, Sara; Parisio, Eva; Boldrini, Enrico; Senesi, Sonia; Campa, Mario

2004-01-01

180

Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population.  

PubMed

This study characterizes the effects of concentrated pomegranate-peel extract (CPE) addition to the TMR at levels of 1, 2, or 4% on voluntary intake, in vivo digestibility, milk yield and composition, and profile of rumen bacterial and archaeal populations in lactating Holstein cows. Supplementation of CPE significantly affected the abundance of methanogenic archaea and specific ruminal bacterial species related to cellulolytic activities and soluble sugar and lactic acid fermentation, as revealed by real-time PCR quantification. Furthermore, CPE supplementation had a significant dose-dependent effect on the whole ruminal bacterial community, as determined by automated ribosomal intergenic spacer analysis. These changes were accompanied by a significant increase in digestibility of dry matter, crude protein, and neutral detergent fiber, as well as milk and energy-corrected milk yields in cows fed the 4% CPE supplement. These results suggest that CPE supplementation significantly affects the rumen bacterial communities, which in turn may be related to a beneficial effect on dairy cow performance. PMID:22863105

Jami, E; Shabtay, A; Nikbachat, M; Yosef, E; Miron, J; Mizrahi, I

2012-08-03

181

A mucoadhesive polymer extracted from tamarind seed improves the intraocular penetration and efficacy of rufloxacin in topical treatment of experimental bacterial keratitis.  

PubMed

Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization. In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental Pseudomonas aeruginosa and Staphylococcus aureus keratitis in rabbits. The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes. Rufloxacin delivered by the polysaccharide reduced P. aeruginosa and S. aureus in the cornea at a higher rate than that obtained by rufloxacin alone. In particular, use of the polysaccharide allowed a substantial reduction of S. aureus in the cornea to be achieved even when the time interval between drug administrations was extended. These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs. The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis. PMID:15328102

Ghelardi, Emilia; Tavanti, Arianna; Davini, Paola; Celandroni, Francesco; Salvetti, Sara; Parisio, Eva; Boldrini, Enrico; Senesi, Sonia; Campa, Mario

2004-09-01

182

Extracting single genomes from heterogenous DNA samples: A test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta)  

PubMed Central

Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.

Dale, Colin; Dunbar, Helen; Moran, Nancy A.; Ochman, Howard

2005-01-01

183

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis  

Microsoft Academic Search

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analy- sis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to pro- vide a more consistent higher quality alignment and faster processing of user sequences. Substan- tial new analysis features include a new Pyrose- quencing Pipeline that provides tools

James R. Cole; Q. Wang; E. Cardenas; J. Fish; B. Chai; Ryan J. Farris; A. S. Kulam-syed-mohideen; D. M. Mcgarrell; Terry L. Marsh; George M. Garrity; James M. Tiedje

2009-01-01

184

Antimicrobial effects of aqueous plant extracts on the fungi Microsporum canis and Trichophyton rubrum and on three bacterial species.  

PubMed

Aqueous extracts of 10 plants were tested for their ability to inhibit Trichophyton rubrum and Microsporum canis, the aetiological agents of dermal fungal infections in humans. These extracts were also evaluated for their activity against some bacteria. Aqueous extracts from the leaves of Inula viscosa produced detectable antifungal activity against these dermatophytes. PMID:9489036

Maoz, M; Neeman, I

1998-01-01

185

Analyses of bacterial communities in meju, a Korean traditional fermented soybean bricks, by cultivation-based and pyrosequencing methods.  

PubMed

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before. PMID:21717316

Kim, Yi-Seul; Kim, Min-Cheol; Kwon, Soon-Wo; Kim, Soo-Jin; Park, In-Cheol; Ka, Jong-Ok; Weon, Hang-Yeon

2011-06-30

186

Diagnosis of fulminant pneumonia caused by Legionella pneumophila serogroup 8 with the sequence analysis of the 16S rRNA gene.  

PubMed

Pneumonia is the fourth leading cause of death in Japan. Accurate and rapid detection of the causative pathogen(s) is necessary and important for appropriate antimicrobial treatment, especially in patients with rapidly progressive pneumonia or immunocompromised patients. Conventional methods, such as cultivations, detection of urinary antigens or PCR amplification of specific genes, inevitably require the precise presumption of potential pathogens in each case, and pneumonia caused by unanticipated microorganisms might lead to inadequate antimicrobial treatments and unfortunate consequences. We herein report an immunocompromised female patient (69 years old) with fulminant pneumonia caused by Legionella (L.) pneumophila serogroup 8. Ordinary cultivation methods and urinary antigen detection failed to identify the causative organisms. Accordingly, DNA was extracted from the bronchoalveolar lavage fluid and used for the PCR-based cloning of the bacterial 16S rRNA gene. Sequencing analysis of the isolated clones revealed the predominance of L. pneumophila. Based on this information, the patient received an appropriate and successful antimicrobial treatment. In addition, L. pneumophila serogroup 8 was identified with culturing the bronchoalveolar lavage fluid and serotyping with L. pneumophila antisera. The 16S rRNA gene sequencing analysis can reveal the potential pathogens without any presumption about the organism, and can evaluate the kinds and ratio of bacterial species in each specimen. In conclusion, this cultivation-independent method is a potential diagnostic modality for pneumonia, especially in patients with rapidly progressive pneumonia or those who are immunocompromised. PMID:21878746

Kawanami, Toshinori; Yatera, Kazuhiro; Fukuda, Kazumasa; Yamasaki, Kei; Kunimoto, Masamizu; Nagata, Shuya; Nishida, Chinatsu; Ishimoto, Hiroshi; Ogawa, Midori; Taniguchi, Hatsumi; Mukae, Hiroshi

2011-01-01

187

Benthic bacterial diversity in submerged sinkhole ecosystems.  

PubMed

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

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

2009-10-30

188

Bacterial diversity in soil enrichment cultures amended with 2 (2-methyl-4-chlorophenoxy) propionic acid (mecoprop).  

PubMed

Summary The tfdA gene encodes for an alpha-ketoglutarate-dependent dioxygenase enzyme which catalyses the first step of the degradation of phenoxyalkanoic acid herbicides such as 2 (2-methyl-4-chlorophenoxy) propionic acid (mecoprop). The bacterial diversity of soil enrichment cultures containing mecoprop was examined by Denaturing Gradient Gel Electrophoresis (DGGE) and clone libraries of both 16S rRNA genes and tfdA genes. The 16S rRNA gene sequences were diverse and clustered with either the Beta- or Gammaproteobacteria. The 16S rRNA gene sequence from a bacterial strain isolated from an enrichment culture, grown on R-mecoprop, which represented a dominant band in the DGGE profiles, had a high 16S rRNA sequence identity (100%) to Burkholderia glathei. This is the first report that B. glathei is implicated in mecoprop degradation. PCR amplification of the tfdA genes detected class III tfdA genes only, and no class I or class II tfdA sequences were detected. To understand the genes involved the degradation of specific mecoprop (R-) and (S-) enantiomers, oligonucleotide probes targeting the tfdA, rdpA, sdpA and cadA genes were hybridized to DNA extracted from enrichment cultures grown on either R-mecoprop or (R/S) racemic mecoprop. Strong hybridization signals were obtained with sdpA and tfdA probes using DNA extracted from cultures grown on racemic mecoprop. A strong hybridization signal was also obtained with the rdpA probe with DNA extracted from the cultures grown on R-mecoprop. This suggests the rdpA gene is involved in R-mecoprop degradation while tfdA, sdpA and cadA genes are involved in the degradation of both R- and S-mecoprop. PMID:17803781

Zakaria, Dalia; Lappin-Scott, Hilary; Burton, Sara; Whitby, Corinne

2007-10-01

189

Large Variations in Bacterial Ribosomal RNA Genes  

PubMed Central

Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti–Shine-Dalgarno sequence (5?-CCTCC-3?). This loss was accompanied by elimination of Shine-Dalgarno–like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery.

Lim, Kyungtaek; Furuta, Yoshikazu; Kobayashi, Ichizo

2012-01-01

190

Large variations in bacterial ribosomal RNA genes.  

PubMed

Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti-Shine-Dalgarno sequence (5'-CCTCC-3'). This loss was accompanied by elimination of Shine-Dalgarno-like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery. PMID:22446745

Lim, Kyungtaek; Furuta, Yoshikazu; Kobayashi, Ichizo

2012-03-24

191

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis  

Microsoft Academic Search

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic frame- work for these data. Updated monthly, these services are made available through the RDP-II website (http:\\/\\/ rdp.cme.msu.edu\\/). RDP-II release 9.21 (August 2004) contains 101632 bacterial small subunit rRNA gene sequences in aligned and annotated

James R. Cole; B. Chai; Ryan J. Farris; Q. Wang; S. A. Kulam; D. M. Mcgarrell; George M. Garrity; James M. Tiedje

2005-01-01

192

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods  

PubMed Central

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

Loonen, A. J. M.; Jansz, A. R.; Kreeftenberg, H.; Bruggeman, C. A.; Wolffs, P. F. G.

2010-01-01

193

Statistical optimization of medium composition for bacterial cellulose production by Gluconacetobacter hansenii UAC09 using coffee cherry husk extract--an agro-industry waste.  

PubMed

During the production of grape wine, the formation of thick leathery pellicle/bacterial cellulose (BC) at the airliquid interface was due to the bacterium, which was isolated and identified as Gluconacetobacter hansenii UAC09. Cultural conditions for bacterial cellulose production from G. hansenii UAC09 were optimized by central composite rotatable experimental design. To economize the BC production, coffee cherry husk (CCH) extract and corn steep liquor (CSL) were used as less expensive sources of carbon and nitrogen, respectively. CCH and CSL are byproducts from the coffee processing and starch processing industry, respectively. The interactions between pH (4.5- 8.5), CSL (2-10%), alcohol (0.5-2%), acetic acid (0.5- 2%), and water dilution rate to CCH ratio (1:1 to 1:5) were studied using response surface methodology. The optimum conditions for maximum BC production were pH (6.64), CSL (10%), alcohol (0.5%), acetic acid (1.13%), and water to CCH ratio (1:1). After 2 weeks of fermentation, the amount of BC produced was 6.24 g/l. This yield was comparable to the predicted value of 6.09 g/l. This is the first report on the optimization of the fermentation medium by RSM using CCH extract as the carbon source for BC production by G. hansenii UAC09. PMID:21791961

Rani, Mahadevaswamy Usha; Rastogi, Navin K; Appaiah, K A Anu

2011-07-01

194

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil  

Microsoft Academic Search

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3)

K. Hjort; M. Bergstrom; M. F. Adesina; J. K. Jansson; K. Smalla; S. Sjoling

2009-01-01

195

Bacterial clearance, heterophil function, and hematological parameters of transport stressed turkey poults supplemented with dietary yeast extract  

Technology Transfer Automated Retrieval System (TEKTRAN)

Yeast extracts contain biological response modifiers that may be useful as alternatives to antibiotics for controlling pathogens in poultry production and mitigating the deleterious effects of production stressors. A standardized yeast extract feed supplement, Alphamune™ (YE), was added to turkey po...

196

Extract of corn silk (stigma of Zea mays) inhibits the tumour necrosis factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1 expression.  

PubMed

Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking. PMID:9619111

Habtemariam, S

1998-05-01

197

Characterization of soil bacterial communities in rhizospheric and nonrhizospheric soil of Panax ginseng.  

PubMed

A culture-independent approach was used to evaluate the bacterial community in rhizospheric and nonrhizospheric soil in which Panax ginseng had grown for 3 years. For each sample, soil was randomly collected from multiple sampling points and mixed thoroughly before genomic DNA extraction. Universal primers 27f and 1492r were used to amplify 16S rRNA genes. Clone libraries were constructed using the amplified 16S rRNA genes, and 192 white clones were chosen for further sequencing. After digestion with restriction endonuclease, 44 operational taxonomic units (OTUs) were generated for rhizospheric and 21 OTUs for nonrhizospheric soils, and the clones of each OTU were sequenced. Blast analysis showed that bacillus, acidobacteria, and proteobacteria were the dominant populations in rhizospheric soil, and proteobacteria were dominant in nonrhizospheric soil. Phylogenetic results showed that bacillus and acidobacteria were clustered into the group of uncultured bacteria in rhizospheric soil; however, proteobacteria were the unique dominant in nonrhizospheric soil. PMID:22875735

Ying, Yi Xin; Ding, Wan Long; Li, Yong

2012-08-09

198

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR  

Microsoft Academic Search

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces\\u000a is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial\\u000a contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps\\u000a of sampling and sample processing. We used quantitative real-time

S. P. van Tongeren; J. E. Degener; H. J. M. Harmsen

199

Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.  

PubMed

The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum. PMID:22538976

Corrigan, A; Horgan, K; Clipson, N; Murphy, R A

2012-04-27

200

Taxonomy of bacterial fish pathogens  

PubMed Central

Bacterial taxonomy has progressed from reliance on highly artificial culture-dependent techniques involving the study of phenotype (including morphological, biochemical and physiological data) to the modern applications of molecular biology, most recently 16S rRNA gene sequencing, which gives an insight into evolutionary pathways (= phylogenetics). The latter is applicable to culture-independent approaches, and has led directly to the recognition of new uncultured bacterial groups, i.e. "Candidatus", which have been associated as the cause of some fish diseases, including rainbow trout summer enteritic syndrome. One immediate benefit is that 16S rRNA gene sequencing has led to increased confidence in the accuracy of names allocated to bacterial pathogens. This is in marked contrast to the previous dominance of phenotyping, and identifications, which have been subsequently challenged in the light of 16S rRNA gene sequencing. To date, there has been some fluidity over the names of bacterial fish pathogens, with some, for example Vibrio anguillarum, being divided into two separate entities (V. anguillarum and V. ordalii). Others have been combined, for example V. carchariae, V. harveyi and V. trachuri as V. harveyi. Confusion may result with some organisms recognized by more than one name; V. anguillarum was reclassified as Beneckea and Listonella, with Vibrio and Listonella persisting in the scientific literature. Notwithstanding, modern methods have permitted real progress in the understanding of the taxonomic relationships of many bacterial fish pathogens.

2011-01-01

201

Compilation of 5S rRNA and 5S rRNA gene sequences  

PubMed Central

This is an update for the 5S rRNA sequences of the BERLIN RNA DATABANK last published in 1990 (1). The new entry consists of 25 eubacterial and 2 eukaryotic 5S rRNA sequences and 10 plant 5S rRNA pseudogenes (Table 1). Thus the BERLIN RNA DATABANK contains as of Febuary 1, 1991 the 5S rRNA sequences of 44 archaebacteria, 292 eubacteria, 20 plastids, 6 mitochondria, 321 eukaryotes and 21 eukaryotic pseudogenes. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information.

Specht, Thomas; Wolters, Jorn; Erdmann, Volker A.

1991-01-01

202

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods.  

PubMed

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M(Grade)), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay. PMID:20972809

Loonen, A J M; Jansz, A R; Kreeftenberg, H; Bruggeman, C A; Wolffs, P F G; van den Brule, A J C

2010-10-24

203

Microencapsulation of Cheese Ripening Systems: Production of Diacetyl and Acetoin in Cheese by Encapsulated Bacterial Cell-Free Extract  

Microsoft Academic Search

A ceil-free extract of Streptococcus lactis subsp, diacetilactis was encapsulated with substrates and cofactors for gen- eration of diacetyl and acetoin. This model system was used to demonstrate the feasibility of incorporating milk fat-coated microcapsules containing se- lected enzymes and substrates into cheese to enhance and regulate flavor develop- ment. Concentrations of diacetyl and acetoin in cheese containing encapsulated enzymes

E. L. Magee Jr.; N. F. Olson; R. C. Lindsay

1981-01-01

204

Bacterial diversity in hyperarid Atacama Desert soils  

NASA Astrophysics Data System (ADS)

Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (aw) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 ?g/g < TOC < 1000 ?g/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 × 105 to 7 × 106 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

Connon, Stephanie A.; Lester, Elizabeth D.; Shafaat, Hannah S.; Obenhuber, Donald C.; Ponce, Adrian

2007-12-01

205

Multiple rRNA variants in a single spore of the microsporidian Nosema bombi.  

PubMed

To understand the source of the multiple DNA sequence variants of Nosema bombi ribosomal RNA (rRNA) found in a single bumble bee host, we PCR amplified, cloned, and sequenced the partial rRNA gene from 125 clones, which were derived from four out of 46 spores individually isolated from a single host by laser microdissection. At least two rRNA variants, characterized by either (GTTT)(2) or (GTTT)(3) repeat units within the internal transcribed spacer (ITS) region, were found per spore in approximately equal proportions, variants which were also found in approximately equal proportions in 55 clones of the two DNA extracts of multiple spores from the same host. Firstly, we demonstrate for the first time that DNA sequences can be obtained from single-binucleate microsporidia. Secondly, it appears that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore or even within a single nucleus. We thereby demonstrate unequivocally that two or more rRNA sequence variants exist per N. bombi spore, and urge caution in the use of multicopy rRNA genes for population genetic and phylogenetic analysis of this and other Microsporidia unless homologous copies can be reliably typed. PMID:17300528

O'Mahony, Elaine M; Tay, Wee Tek; Paxton, Robert J

206

Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes? †  

PubMed Central

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

Pei, Anna Y.; Oberdorf, William E.; Nossa, Carlos W.; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A.; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M.; Sotero, Steven; DeSantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

2010-01-01

207

Diversity of 16S rRNA genes within individual prokaryotic genomes.  

PubMed

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases. PMID:20418441

Pei, Anna Y; Oberdorf, William E; Nossa, Carlos W; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M; Sotero, Steven; Desantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

2010-04-23

208

Structural basis for ribosomal 16S rRNA cleavage by the cytotoxic domain of colicin E3  

PubMed Central

The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves 16S rRNA between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3 that activate the 16S rRNA for 2? OH induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and Helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.

Meenan, Nicola AG; Sharma, Amit; Kelley, Ann C; Kleanthous, Colin; Ramakrishnan, V

2013-01-01

209

Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining  

Microsoft Academic Search

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to afluorescently labeled universal 16S rRNA probe; 3H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4*,6-diamidino-2-phenylindole) staining;

MARKUS KARNER; ANDJED A. FUHRMAN

1997-01-01

210

Role of the 5.8S rRNA in ribosome translocation.  

PubMed Central

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.

Abou Elela, S; Nazar, R N

1997-01-01

211

5S rRNA Data Bank.  

PubMed Central

In this paper we present the updated version of the compilation of 5S rRNA and 5S rDNA nucleotide sequences. It contains 1622 primary structures of 5S rRNAs and 5S rRNA genes from 888 species. These include 58 archaeal, 427 eubacterial, 34 plastid, nine mitochondrial and 1094 eukaryotic DNA or RNA nucleotide sequences. The sequence entries are divided according to the taxonomic position of the organisms. All individual sequences deposited in the 5S rRNA Database can be retrieved using the WWW-based, taxonomic browser at http://rose.man.poznan.pl/5SData/5SRNA.html++ + or http://www.chemie. fu-berlin.de/fb_chemie/agerdmann/5S_rRNA.html . The files with complete sets of data as well as sequence alignments are available via anonymous ftp.

Szymanski, M; Specht, T; Barciszewska, M Z; Barciszewski, J; Erdmann, V A

1998-01-01

212

Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis.  

PubMed

Investigation of the initial and spoilage microbial diversity of iced stored sea bream was carried out. Culture dependent methods were used for bacterial enumeration and phenotypic identification of bacterial isolates, while culture independent methods, using bacterial 16S rRNA gene amplification, cloning and sequencing of DNA extracted directly from the flesh were also employed. The culture dependent approach revealed that the initial microbiota was dominated by Acinetobacter, Shewanella, Pseudomonas and Flavobacterium, while at the end of shelf-life determined by sensory analysis (16 days), the predominant microbiota was Pseudomonas and Shewanella. Culture independent approach showed that initially the sea bream flesh was strongly dominated by Acinetobacter, while Pseudomonas, Aeromonas salmonicida and Shewanella were the predominant phylotypes at the end of shelf-life. Initial and spoilage microbiota comprised of phylotypes previously identified by others using traditional or molecular techniques. However, Aeromonas has not been reported as part of the dominant microbiota of sea bream at the time of spoilage. Combination of classical and molecular methodologies better reveals the microbiota during storage by revealing bacteria that escape standard approaches and, thus, provides valuable complementary information regarding microbiological spoilage. PMID:23122505

Parlapani, F F; Meziti, A; Kormas, K Ar; Boziaris, I S

2012-09-13

213

Serenoa repens associated with Urtica dioica (ProstaMEV ®) and curcumin and quercitin (FlogMEV ®) extracts are able to improve the efficacy of prulifloxacin in bacterial prostatitis patients: results from a prospective randomised study  

Microsoft Academic Search

We report the results of a prospective randomised study to evaluate the therapeutic effect of Serenoa repens, Urtica dioica (ProstaMEV®), quercitin and curcumin (FlogMEV®) extracts associated with prulifloxacin in patients affected by chronic bacterial prostatitis (CBP). From a whole population of 284 patients, 143 patients affected by CBP [National Institutes of Health (NIH) class II prostatitis] were enrolled. All patients

Tommaso Cai; Sandra Mazzoli; Adriano Bechi; Patrizia Addonisio; Nicola Mondaini; Roberto Castricchi Pagliai; Riccardo Bartoletti

2009-01-01

214

The intestinal bacterial community in the food waste-reducing larvae of Hermetia illucens.  

PubMed

As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds. PMID:21267722

Jeon, Hyunbum; Park, Soyoung; Choi, Jiyoung; Jeong, Gilsang; Lee, Sang-Beom; Choi, Youngcheol; Lee, Sung-Jae

2011-01-26

215

Characterization of Microbial Diversity by Determining Terminal Restriction Fragment Length Polymorphisms of Genes Encoding 16S rRNA  

Microsoft Academic Search

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5* end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction

WEN-TSO LIU; TERENCE L. MARSH; HANS CHENG; LARRY J. FORNEY

1997-01-01

216

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques  

Microsoft Academic Search

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34\\/38 (89.5%) of total bacterial numbers consisting of

Mayumi Murayama; Yuki Kakinuma; Yasunori Maeda; Juluri R. Rao; Motoo Matsuda; Jiru Xu; Peter JA Moore; B. Cherie Millar; Paul J. Rooney; Colin E. Goldsmith; Anne Loughrey; M. Ann S. McMahon; David A. McDowell; John E. Moore

2010-01-01

217

Culture-independent analysis of bacterial diversity in a child-care facility  

PubMed Central

Background Child-care facilities appear to provide daily opportunities for exposure and transmission of bacteria and viruses. However, almost nothing is known about the diversity of microbial contamination in daycare facilities or its public health implications. Recent culture-independent molecular studies of bacterial diversity in indoor environments have revealed an astonishing diversity of microorganisms, including opportunistic pathogens and many uncultured bacteria. In this study, we used culture and culture-independent methods to determine the viability and diversity of bacteria in a child-care center over a six-month period. Results We sampled surface contamination on toys and furniture using sterile cotton swabs in four daycare classrooms. Bacteria were isolated on nutrient and blood agar plates, and 16S rRNA gene sequences were obtained from unique (one of a kind) colony morphologies for species identification. We also extracted DNA directly from nine representative swab samples taken over the course of the study from both toy and furniture surfaces, and used "universal" 16S rRNA gene bacterial primers to create PCR-based clone libraries. The rRNA gene clones were sequenced, and the sequences were compared with related sequences in GenBank and subjected to phylogenetic analyses to determine their evolutionary relationships. Culturing methods identified viable bacteria on all toys and furniture surfaces sampled in the study. Bacillus spp. were the most commonly cultured bacteria, followed by Staphylococcus spp., and Microbacterium spp. Culture-independent methods based on 16S rRNA gene sequencing, on the other hand, revealed an entirely new dimension of microbial diversity, including an estimated 190 bacterial species from 15 bacterial divisions. Sequence comparisons and phylogenetic analyses determined that the clone libraries were dominated by a diverse set of sequences related to Pseudomonas spp., as well as uncultured bacteria originally identified on human vaginal epithelium. Other sequences were related to uncultured bacteria from wastewater sludge, and many human-associated bacteria including a number of pathogens and opportunistic pathogens. Our results suggest that the child-care facility provided an excellent habitat for slime-producing Pseudomonads, and that diaper changing contributed significantly to the bacterial contamination. Conclusion The combination of culture and culture-independent methods provided powerful means for determining both viability and diversity of bacteria in child-care facilities. Our results provided insight into the source of contamination and suggested ways in which sanitation might be improved. Although our study identified a remarkable array of microbial diversity present in a single daycare, it also revealed just how little we comprehend the true extent of microbial diversity in daycare centers or other indoor environments.

2007-01-01

218

A Mucoadhesive Polymer Extracted from Tamarind Seed Improves the Intraocular Penetration and Efficacy of Rufloxacin in Topical Treatment of Experimental Bacterial Keratitis  

Microsoft Academic Search

Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concen- trated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to

Emilia Ghelardi; Arianna Tavanti; Paola Davini; Francesco Celandroni; Sara Salvetti; Eva Parisio; Enrico Boldrini; Sonia Senesi; Mario Campa

2004-01-01

219

Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.  

PubMed

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water. PMID:21845446

Kahlisch, Leila; Henne, Karsten; Gröbe, Lothar; Brettar, Ingrid; Höfle, Manfred G

2011-08-16

220

Direct PCR amplification of the 16S rRNA gene from single microbial cells isolated from an Antarctic iceberg using laser microdissection microscopy  

NASA Astrophysics Data System (ADS)

Here, we describe a technique that allows the genetic linage analysis of 16S rRNA genes in bacteria observed under a microscope. The technique includes the isolation of microbial cells using a laser microdissection microscope, lysis of the cells, and amplification of the 16S rRNA genes in the isolated cells without interference by bacterial DNA contamination from the experimental environment or reagents. Using this technique, we successfully determined 15 16S rRNA gene sequences in cells isolated from an Antarctic iceberg. These sequences showed 94%-100% identity to their closest strains, which included bacteria that occur in aqueous, marine, and soil environments.

Yanagihara, Katsuhiko; Niki, Hironori; Baba, Tomoya

2011-09-01

221

Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography  

PubMed Central

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

Ginige, Maneesha P.; Keller, Jurg; Blackall, Linda L.

2005-01-01

222

Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases  

PubMed Central

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

Clarridge, Jill E.

2004-01-01

223

Eukaryotic 5S rRNA biogenesis  

PubMed Central

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function.

Ciganda, Martin; Williams, Noreen

2012-01-01

224

Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.  

PubMed Central

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.

Taylor, D E; Ge, Z; Purych, D; Lo, T; Hiratsuka, K

1997-01-01

225

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil.  

PubMed

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples. PMID:23974369

Han, Ping; Li, Meng; Gu, Ji-Dong

2013-08-24

226

Polyphasic characterization of bacterial community in fresh cut salads.  

PubMed

In the present work we describe a polyphasic study of bacterial community in fresh cut salads packaged under ordinary and modified atmospheres. Samples of fresh cut salads were aseptically collected at 0, 3, and 6 days of storage and analysed both by culture-dependent and -independent methods. DNA extracted from fresh cut salad samples was used as a template for PCR amplification of 16 S rRNA gene; the PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE); finally, clone libraries of 16 S rRNA gene from the fresh cut salad was constructed. Results of plating count revealed a significant increase of all microbial loads in fresh-cut salad samples packaged in OA and that the microbial growth of the different groups was significantly affected by the conditions applied for MA packaging. A constant presence, throughout storage, of the pathogenic bacteria in all the fresh-cut salads samples was highlighted by PCR-DGGE analysis. Therefore, the polyphasic approach used in the present study allowed us to characterize the main species involved in the fresh cut salad products and to better understand their dynamics throughout storage. PMID:19027975

Randazzo, Cinzia Lucia; Scifò, Giovanna Ombretta; Tomaselli, Filippo; Caggia, Cinzia

2008-10-29

227

Bacterial diversity in two Neonatal Intensive Care Units (NICUs).  

PubMed

Infants in Neonatal Intensive Care Units (NICUs) are particularly susceptible to opportunistic infection. Infected infants have high mortality rates, and survivors often suffer life-long neurological disorders. The causes of many NICU infections go undiagnosed, and there is debate as to the importance of inanimate hospital environments (IHEs) in the spread of infections. We used culture-independent next-generation sequencing to survey bacterial diversity in two San Diego NICUs and to track the sources of microbes in these environments. Thirty IHE samples were collected from two Level-Three NICU facilities. We extracted DNA from these samples and amplified the bacterial small subunit (16S) ribosomal RNA gene sequence using 'universal' barcoded primers. The purified PCR products were pooled into a single reaction for pyrosequencing, and the data were analyzed using QIIME. On average, we detected 93+/-39 (mean +/- standard deviation) bacterial genera per sample in NICU IHEs. Many of the bacterial genera included known opportunistic pathogens, and many were skin-associated (e.g., Propionibacterium). In one NICU, we also detected fecal coliform bacteria (Enterobacteriales) in a high proportion of the surface samples. Comparison of these NICU-derived sequences to previously published high-throughput 16S rRNA amplicon studies of other indoor environments (offices, restrooms and healthcare facilities), as well as human- and soil-associated environments, found the majority of the NICU samples to be similar to typical building surface and air samples, with the notable exception of the IHEs which were dominated by Enterobacteriaceae. Our findings provide evidence that NICU IHEs harbor a high diversity of human-associated bacteria and demonstrate the potential utility of molecular methods for identifying and tracking bacterial diversity in NICUs. PMID:23372757

Hewitt, Krissi M; Mannino, Frank L; Gonzalez, Antonio; Chase, John H; Caporaso, J Gregory; Knight, Rob; Kelley, Scott T

2013-01-23

228

Bacterial Diversity in Two Neonatal Intensive Care Units (NICUs)  

PubMed Central

Infants in Neonatal Intensive Care Units (NICUs) are particularly susceptible to opportunistic infection. Infected infants have high mortality rates, and survivors often suffer life-long neurological disorders. The causes of many NICU infections go undiagnosed, and there is debate as to the importance of inanimate hospital environments (IHEs) in the spread of infections. We used culture-independent next-generation sequencing to survey bacterial diversity in two San Diego NICUs and to track the sources of microbes in these environments. Thirty IHE samples were collected from two Level-Three NICU facilities. We extracted DNA from these samples and amplified the bacterial small subunit (16S) ribosomal RNA gene sequence using ‘universal’ barcoded primers. The purified PCR products were pooled into a single reaction for pyrosequencing, and the data were analyzed using QIIME. On average, we detected 93+/?39 (mean +/? standard deviation) bacterial genera per sample in NICU IHEs. Many of the bacterial genera included known opportunistic pathogens, and many were skin-associated (e.g., Propionibacterium). In one NICU, we also detected fecal coliform bacteria (Enterobacteriales) in a high proportion of the surface samples. Comparison of these NICU-derived sequences to previously published high-throughput 16S rRNA amplicon studies of other indoor environments (offices, restrooms and healthcare facilities), as well as human- and soil-associated environments, found the majority of the NICU samples to be similar to typical building surface and air samples, with the notable exception of the IHEs which were dominated by Enterobacteriaceae. Our findings provide evidence that NICU IHEs harbor a high diversity of human-associated bacteria and demonstrate the potential utility of molecular methods for identifying and tracking bacterial diversity in NICUs.

Hewitt, Krissi M.; Mannino, Frank L.; Gonzalez, Antonio; Chase, John H.; Caporaso, J. Gregory; Knight, Rob; Kelley, Scott T.

2013-01-01

229

Molecular Analysis of Oral and Respiratory Bacterial Species Associated with Ventilator-Associated Pneumonia?  

PubMed Central

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into Escherichia coli, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g., Dialister pneumosintes, Haemophilus segnis, Gemella morbillorum, and Pseudomonas fluorescens) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests.

Bahrani-Mougeot, Farah K.; Paster, Bruce J.; Coleman, Shirley; Barbuto, Sara; Brennan, Michael T.; Noll, Jenene; Kennedy, Thomas; Fox, Philip C.; Lockhart, Peter B.

2007-01-01

230

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability  

Microsoft Academic Search

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fun- gal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal tran- scribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified

L. Ranjard; F. Poly; J.-C. Lata; C. Mougel; J. Thioulouse; S. Nazaret

2001-01-01

231

Increased 5S rRNA oxidation in Alzheimer's disease.  

PubMed

It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD. PMID:22232003

Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

2012-01-01

232

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

PubMed Central

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

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

2011-01-01

233

Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons.  

PubMed

The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of sponge-associated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high- and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms. PMID:20054355

Kamke, Janine; Taylor, Michael W; Schmitt, Susanne

2010-01-07

234

Analysis of the Precursor rRNA Fractions of Rapidly Growing Mycobacteria: Quantification by Methods That Include the Use of a Promoter (rrnA P1) as a Novel Standard†  

PubMed Central

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M. fortuitum infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.

Menendez, Maria del Carmen; Rebollo, Maria Jose; Nunez, Maria del Carmen; Cox, Robert A.; Garcia, Maria Jesus

2005-01-01

235

Common 5S rRNA variants are likely to be accepted in many sequence contexts.  

PubMed

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region. PMID:12569424

Zhang, Zhengdong; D'Souza, Lisa M; Lee, Youn-Hyung; Fox, George E

2003-01-01

236

Bacterial Fertilizers.  

National Technical Information Service (NTIS)

The term bacterial (microbial) fertilizers refers to preparations containing primarily active strains of the microorganisms mainly bacteria in sufficient numbers. This report covers various aspects of bacterial fertilizers: Nitrogen Preparation and Usage;...

W. V. B. Sundra Rao

1981-01-01

237

Analysis of the intestinal microflora in Hepialus gonggaensis larvae Using 16S rRNA sequences.  

PubMed

Gut microbial diversity provides insight into the basic function of a gut microbial ecosystem. In this study, restriction fragment length polymorphism 16S rRNA sequences was used to detect the intestinal microbial diversity of Hepialus gonggaensis larvae. The total DNA of microorganisms was extracted from the intestinal contents and 16S rRNA was amplified. A nearly full-length of 16S rRNA sequence library was constructed. The fingerprints of the microorganisms were analyzed by isolating plasmid and then digesting them with EcoRI, MspI, and HaeIII enzymes, respectively. The library established includes 35 restriction endonuclease types and a phylogenetic tree depicted the linkage of the isolated microbial from the guts of H. gonggaensis larvae. The dominant bacteria in the guts of H. gonggaensis larvae belong to Rahnella sp and Carnobacterium sp and accounted for 45.58% and 30.88% of the total 16S rRNA clones library, respectively. The result showed that bacteria diversity in the guts of H. gonggaensis larvae had some differences from those isolated from normal environment. PMID:18183461

Yu, Hewei; Wang, Zhongkang; Liu, Li; Xia, Yuxian; Cao, Yueqing; Yin, Youping

2008-01-09

238

Molecular bacterial diversity of a forest soil under residue management regimes in subtropical Australia.  

PubMed

A major operational change in exotic pine plantations of subtropical Australia has been the decision to retain postharvest residues on site. A long-term field experiment was established in February 1996 to examine the impacts of residue management regimes [i.e. the postharvest residues removed (G0R), natural amount of residues retained (G1R) and residue quantity doubled and retained (G2R)] on tree growth (F1 hybrid pine) and sustainable soil management. Twelve soil samples, which included the above three residue regimes with four replicates, were collected at plantation age 6.4 years. A 16S rRNA gene clone library was established following soil community DNA extraction, polymerase chain reaction amplification and cloning. A total of 324 clones, including 27 from each sample, were randomly selected and sequenced to represent the bacterial composition and diversity of the clone library and thus the soil bacterial community under the residue management regimes. Phylogenetic analyses indicated that Acidobacteria (37.6%) and Proteobacteria (35.6%) were the dominant components of the soil bacterial community, followed by Actinobacteria (14.7%), Chlamydiae/Verrucomicrobia (7.3%), Unclassified Bacteria (3.8%) and Gemmatimonadetes (1.0%). Analysis of molecular variance revealed that there was no significant difference in bacterial composition and diversity among the residue management regimes or their replicated samples. PMID:16420613

He, Jizheng; Xu, Zhihong; Hughes, Jane

2006-01-01

239

Non-Watson-Crick Basepairing and Hydration in RNA Motifs: Molecular Dynamics of 5S rRNA Loop E  

Microsoft Academic Search

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries

Kamila Réblová; Nad’a Špa?ková; Richard Štefl; Kristina Csaszar; Jaroslav Ko?a; Neocles B. Leontis; Ji?í Šponer

2003-01-01

240

Clostridia dominate 16S rRNA gene libraries prepared from the hindgut of temperate marine herbivorous fishes  

Microsoft Academic Search

Bacterial diversity in the microbial communities of posterior gut sections of three temperate marine herbivorous fish species\\u000a from New Zealand was characterised using Amplified Ribosomal DNA Restriction Analysis, and 16S rRNA gene amplification and\\u000a sequencing methods. The fish were collected in 1999–2000 in the Hauraki Gulf, New Zealand (35°54’–36°24’S, 174°48’–175°25’E).\\u000a The gastrointestinal bacterial communities of Kyphosus sydneyanus (Günther, 1886) (F.

Kendall D. Clements; Isabel B. Y. Pasch; Damian Moran; Susan J. Turner

2007-01-01

241

Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms  

PubMed Central

The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13.

Hochrein, Hubertus; Kirschning, Carsten J.

2013-01-01

242

16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting  

PubMed Central

Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases.

Navarro-Noya, Yendi; Hernandez-Rodriguez, Cesar; Zenteno, Juan C.; Buentello-Volante, Beatriz; Cancino-Diaz, Mario E.; Jan-Roblero, Janet; Cancino-Diaz, Juan C.

2012-01-01

243

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.  

PubMed

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria. PMID:16723545

Aellen, Steve; Que, Yok-Ai; Guignard, Bertrand; Haenni, Marisa; Moreillon, Philippe

2006-06-01

244

Assessment of microbial dynamics in the Pearl River Estuary by 16S rRNA terminal restriction fragment analysis  

NASA Astrophysics Data System (ADS)

We have evaluated the feasibility of using the terminal restriction fragment length polymorphism (T-RFLP) pattern of polymerase chain reaction (PCR) amplified 16S rRNA sequences to track the changes of the free-living bacterial community for the Pearl River Estuary surface waters. The suitability of specific PCR primers, PCR bias induced by thermal cycles, and field-sampling volumes were critically evaluated in laboratory tests. We established a workable protocol and obtained TRF patterns that reflected the changes in the bacterial population. The temporal dynamics over a 24 h period were examined at one anchored station, as well as the spatial distribution pattern of the bacterial community at several stations, covering the transects along the river discharge direction and across the river plume. The TRF pattern revealed 9 dominant bacterial groups. Changes in their relative abundance reflecting the changes in the bacterial community composition were documented. Many culturable species were isolated from each field sample and a portion of the 16S rRNA gene for each species was sequenced. The species was identified based on sequence data comparison. In this region, the dominant species belong to the ?-subdivision of proteobacteria and the Bacillus/Clostridium group of Firmicutes. We also detected the wide spread distribution of Acinetobacter spp.; many of these species are known nosocomial pathogen for humans.

Wu, Madeline; Song, Liansheng; Ren, Jianping; Kan, Jianjun; Qian, Pei-Yuan

2004-10-01

245

Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland  

Microsoft Academic Search

Bacterial diversity in alkaline (pH 10.4) and permanently cold (4°C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was

Mariane Schmidt; Anders Priemé; Peter Stougaard

2006-01-01

246

Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China  

Microsoft Academic Search

Surface soils from Guiyu, China (an intense e-waste processing center) were analyzed for persistent organic pollutants (POPs)\\u000a and variations in composition of the resident bacterial communities. Denaturing Gradient Gel Electrophoresis analysis of bacterial\\u000a 16S rRNA gene showed that e-waste pollution altered the bacterial community structure by promoting changes in species composition\\u000a and species richness. Bacterial diversity was not decreased at

Wen Zhang; Hui Wang; Rui Zhang; Xie-Zhi Yu; Pei-Yuan Qian; M. H. Wong

2010-01-01

247

Succession of Microbial Communities during Hot Composting as Detected by PCR-Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes  

Microsoft Academic Search

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region

SABINE PETERS; STEFANIE KOSCHINSKY; FRANK SCHWIEGER; CHRISTOPH C. TEBBE

2000-01-01

248

High-throughput 16S rRNA gene sequencing reveals alterations of intestinal microbiota in myalgic encephalomyelitis/chronic fatigue syndrome patients.  

PubMed

Human intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Intestinal dysfunction is a frequent complaint in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients, and previous reports suggest that dysbiosis, i.e. the overgrowth of abnormal populations of bacteria in the gut, is linked to the pathogenesis of the disease. We used high-throughput 16S rRNA gene sequencing to investigate the presence of specific alterations in the gut microbiota of ME/CFS patients from Belgium and Norway. 43 ME/CFS patients and 36 healthy controls were included in the study. Bacterial DNA was extracted from stool samples, PCR amplification was performed on 16S rRNA gene regions, and PCR amplicons were sequenced using Roche FLX 454 sequencer. The composition of the gut microbiota was found to differ between Belgian controls and Norwegian controls: Norwegians showed higher percentages of specific Firmicutes populations (Roseburia, Holdemania) and lower proportions of most Bacteroidetes genera. A highly significant separation could be achieved between Norwegian controls and Norwegian patients: patients presented increased proportions of Lactonifactor and Alistipes, as well as a decrease in several Firmicutes populations. In Belgian subjects the patient/control separation was less pronounced, however some abnormalities observed in Norwegian patients were also found in Belgian patients. These results show that intestinal microbiota is altered in ME/CFS. High-throughput sequencing is a useful tool to diagnose dysbiosis in patients and could help designing treatments based on gut microbiota modulation (antibiotics, pre and probiotics supplementation). PMID:23791918

Frémont, Marc; Coomans, Danny; Massart, Sebastien; De Meirleir, Kenny

2013-06-19

249

Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome  

PubMed Central

Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

Yuan, Sanqing; Cohen, Dora B.; Ravel, Jacques; Abdo, Zaid; Forney, Larry J.

2012-01-01

250

The Potential of Metatranscriptomics for Identifying Screening Targets for Bacterial Vaginosis  

PubMed Central

Background The ribosomal RNA content of a sample collected from a woman with bacterial vaginosis (BV) was analysed to determine the active microbial community, and to identify potential targets for further screening. Methodology/Principal Findings The sample from the BV patient underwent total RNA extraction, followed by physical subtraction of human rRNA and whole transcriptome amplification. The metatranscriptome was sequenced using Roche 454 titanium chemistry. The bioinformatics pipeline MG-RAST and desktop DNA analysis platforms were utilised to analyse results. Bacteria of the genus Prevotella (predominately P. amnii) constituted 36% of the 16S rRNA reads, followed by Megasphaera (19%), Leptotrichia/Sneathia (8%) and Fusobacterium (8%). Comparison of the abundances of several bacteria to quantitative PCR (qPCR) screening of extracted DNA revealed comparable relative abundances. This suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16S rRNA gene amplicon sequencing. To assess the presence of P. amnii in a larger pool of samples, 90 sexually active women were screened using qPCR. This bacterium was found to be strongly associated with BV (P<0.001, OR 23.3 (95%CI:2.9–190.7)) among the 90 women. Conclusions/Significance This study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. Prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with BV, and was found to be detected at a high concentration by qPCR in 31% of cohort with BV, with an association with both oral and penile-vaginal sex.

Twin, Jimmy; Bradshaw, Catriona S.; Garland, Suzanne M.; Fairley, Christopher K.; Fethers, Katherine; Tabrizi, Sepehr N.

2013-01-01

251

Characterisation of the bacterial community in expressed prostatic secretions from patients with chronic prostatitis/chronic pelvic pain syndrome and infertile men: a preliminary investigation  

PubMed Central

The expressed prostatic secretions (EPSs) of men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), infertile men and normal men were subjected to microbiological study. EPSs were collected from the subjects, which included 26 normal men, 11 infertile patients and 51 CP/CPPS patients. DNA was extracted from each specimen, and the V3 regions of the 16S rRNA genes were amplified using universal bacterial primers. The results showed that the EPS 16S rRNA gene-positive rate in the CP/CPPS and infertile patients was much higher than in the normal men, but without any difference among the three patient groups. The denaturing gradient gel electrophoresis (DGGE) method was used to characterize the EPS bacterial community structure of the prostate fluid from patients with CP/CPPS or infertility issues. Principal component analysis (PCA) and partial least squares (PLS) analyses of PCR-DGGE profiles revealed that the EPS bacterial community structure differed among the three groups. Three bands were identified as the key factors responsible for the discrepancy between CP/CPPS patients and infertile patients (P<0.05). Two bands were identified as priority factors in the discrepancy of category IIIA and category IIIB prostatitis patients (P<0.05). According to this research, the ecological balance of the prostate and low urethra tract, when considered as a microenvironment, might play an important role in the maintenance of a healthy male reproductive tract.

Hou, Dong-Sheng; Long, Wen-Min; Shen, Jian; Zhao, Li-Ping; Pang, Xiao-Yan; Xu, Chen

2012-01-01

252

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.  

PubMed

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life. PMID:23316189

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2013-01-07

253

A mitochondrial rRNA dimethyladenosine methyltransferase in Arabidopsis  

PubMed Central

S-adenosyl-l-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3? end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process.

Richter, Uwe; Kuhn, Kristina; Okada, Sachiko; Brennicke, Axel; Weihe, Andreas; Borner, Thomas

2010-01-01

254

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA  

PubMed Central

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (?1911, ?1915, ?1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.

Scheunemann, Ann E.; Graham, William D.; Vendeix, Franck A. P.; Agris, Paul F.

2010-01-01

255

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis, the most prevalent cause of vaginal discharge in the United States, is characterized microbiologically\\u000a by a shift in the vagina away from a lactobacillus-predominant flora and toward a predominantly anaerobic milieu. The cause\\u000a of bacterial vaginosis is unknown, but the epidemiology of the syndrome suggests that it is sexually associated. Bacterial\\u000a vaginosis has been associated with various complications,

Jane R. Schwebke

2000-01-01

256

Bead Array Direct rRNA Capture Assay (rCapA) for Amplification Free Speciation of Mycobacterium Cultures  

PubMed Central

Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay.

de Ronde, Hans; Gonzalez Alonso, Paula; van Soolingen, Dick; Klatser, Paul R.; Anthony, Richard M.

2012-01-01

257

Seasonal Dynamics of Bacterioplankton Community Structure in a Eutrophic Lake as Determined by 5S rRNA Analysis  

PubMed Central

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plußsee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria. Comparison of partial 5S rRNA sequences allowed the detection of changes of single taxa over time. Overall, the whole bacterioplankton community showed two to eight abundant (>4% of the total 5S rRNA) taxa. A distinctive seasonal succession was observed in the taxonomic structure of this pelagic community. A rather-stable community structure, with seven to eight different taxonomic units, was observed beginning in April during the spring phytoplankton bloom. A strong reduction in this diversity occurred at the beginning of the clear-water phase (early May), when only two to four abundant taxa were observed, with one taxon dominating (up to 72% of the total 5S rRNA). The community structure during summer stagnation (June and July) was characterized by frequent changes of different dominating taxa. During late summer, a dinoflagellate bloom (Ceratium hirudinella) occurred, with Comamonas acidovorans (?-subclass of the class Proteobacteria) becoming the dominant bacterial species (average abundance of 43% of the total 5S rRNA). Finally, the seasonal dynamics of the community structure of bacterioplankton were compared with the abundances of other major groups of the aquatic food web, such as phyto- and zooplankton, revealing that strong grazing pressure by zooplankton can reduce microbial diversity substantially in pelagic environments.

Hofle, Manfred G.; Haas, Heike; Dominik, Katja

1999-01-01

258

Seasonal dynamics of bacterioplankton community structure in a eutrophic lake as determined by 5S rRNA analysis.  

PubMed

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plusssee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria. Comparison of partial 5S rRNA sequences allowed the detection of changes of single taxa over time. Overall, the whole bacterioplankton community showed two to eight abundant (>4% of the total 5S rRNA) taxa. A distinctive seasonal succession was observed in the taxonomic structure of this pelagic community. A rather-stable community structure, with seven to eight different taxonomic units, was observed beginning in April during the spring phytoplankton bloom. A strong reduction in this diversity occurred at the beginning of the clear-water phase (early May), when only two to four abundant taxa were observed, with one taxon dominating (up to 72% of the total 5S rRNA). The community structure during summer stagnation (June and July) was characterized by frequent changes of different dominating taxa. During late summer, a dinoflagellate bloom (Ceratium hirudinella) occurred, with Comamonas acidovorans (beta-subclass of the class Proteobacteria) becoming the dominant bacterial species (average abundance of 43% of the total 5S rRNA). Finally, the seasonal dynamics of the community structure of bacterioplankton were compared with the abundances of other major groups of the aquatic food web, such as phyto- and zooplankton, revealing that strong grazing pressure by zooplankton can reduce microbial diversity substantially in pelagic environments. PMID:10388718

Höfle, M G; Haas, H; Dominik, K

1999-07-01

259

Phylogenetic Diversity and Spatial Distribution of the Microbial Community Associated with the Caribbean Deep-water Sponge Polymastia cf. corticata by 16S rRNA, aprA , and amoA Gene Analysis  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA, aprA, and amoA genes demonstrated that a phylogenetically diverse and complex microbial community was associated with the Caribbean deep-water\\u000a sponge Polymastia cf. corticata Ridley and Dendy, 1887. From the 38 archaeal and bacterial 16S rRNA phylotypes identified, 53% branched into the sponge-specific,\\u000a monophyletic sequence clusters determined by previous studies (considering predominantly

Birte Meyer; Jan Kuever

2008-01-01

260

Diversity rankings among bacterial lineages in soil  

Microsoft Academic Search

We used rarefaction curve analysis and diversity ordering-based approaches to rank the 11 most frequently encountered bacterial lineages in soil according to diversity in 5 previously reported 16S rRNA gene clone libraries derived from agricultural, undisturbed tall grass prairie and forest soils (n=26,140, 28 328, 31 818, 13 001 and 53 533). The Planctomycetes, Firmicutes and the ?-Proteobacteria were consistently

Noha H Youssef; Mostafa S Elshahed; Elshahed

2009-01-01

261

Nonradioactive Method To Study Genetic Profiles of Natural Bacterial Communities by PCR-Single-Strand-Conformation Polymorphism  

Microsoft Academic Search

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262

DONG-HUN LEE; YOUNG-GUN ZO; ANDSANG-JONG KIM

1996-01-01

262

Phenytoin and 5-(p-hydroxyphenyl)-5-phenylhydantoin do not alter the effects of bacterial and amplified plaque extracts on cultures of fibroblasts from normal and overgrown gingivae.  

PubMed

Local irritation of gingival tissue by plaque is among the factors which affect development of gingival overgrowth in patients undergoing chronic phenytoin (PHT) therapy. Variability in the cytotoxicity of plaque components or of plaque substances plus PHT and/or its metabolites toward gingival fibroblasts may relate to whether gingival overgrowth forms in a particular patient. Fibroblasts from healthy and overgrown gingivae were incubated with (a) PHT and its major human metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), (b) microbial and "amplified" plaque extracts, and (c) microbial and "amplified" plaque extracts plus PHT and HPPH. Cell numbers and cell-associated protein were determined for each incubation preparation. A wide range in cytotoxic response to a particular microbial or plaque extract occurred among cell strains. Plaque extracts from different subjects had variable cytotoxicity toward a cell strain. The differences among fibroblast strains in response to an extract and the variability in cytotoxicity of different plaque extracts toward a cell strain were not related to their source from normal or overgrown gingivae. Cell numbers and cell-associated protein were similar for incubation mixtures containing extracts with and without PHT and HPPH. These data do not show differences among cytotoxicity levels of plaque extracts, the response of particular gingival fibroblast strains to plaque components, or interaction between drugs and certain plaque samples which explain development of gingival overgrowth in some subjects receiving chronic PHT therapy. PMID:3476609

Smith, Q T; Hinrichs, J E

1987-08-01

263

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis is the most common cause of vaginitis, affecting over 3 million women in the United States annually. Depopulation of lactobacilli from the normal vaginal flora and overgrowth of Gardnerella vaginalis and other anaerobic species are the presumed etiology. To date, no scientific evidence shows that bacterial vaginosis is a sexually transmitted disease. Malodorous vaginal discharge is the most

Jeff Wang

2000-01-01

264

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences  

PubMed Central

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

2013-01-01

265

Impact of dibenzofuran/dibenzo-p-dioxin amendment on bacterial community from forest soil and ring-hydroxylating dioxygenase gene populations.  

PubMed

The impact of dibenzofuran (DF) and dibenzo-p-dioxin (DD) on the changes in bacterial community structure and the transition of catabolic genes were studied using forest soil. The bacterial community structure of soil suspensions amended with 1 microg/g of either DF or DD was analyzed by 16S rRNA and functional gene sequencing. To analyze the functional genes in the communities, we targeted a gene sequence that functions as the binding site of Rieske iron sulfur center common to ring-hydroxylating dioxygenases (RHDs) for monocyclic, bicyclic, and tricyclic aromatic compounds. The gene fragments were polymerase chain reaction-amplified from DNAs extracted from soil suspensions spiked with either DF or DD, cloned, and sequenced (70 clones). Bacterial community analysis based on 16S rRNA genes revealed that specific 16S rRNA gene sequences, in particular, phylotypes within alpha-Proteobacteria, increased in the soil suspension amended with DF or DD. RHD gene-based functional community analysis showed that, in addition to two groups of RHD genes that were also detected in unamended soil suspensions, another two groups of RHD genes, each of which is specific to DF- and DD-amended soil, respectively, emerged to a great extent. The DD-specific genotype is phylogenetically distant from any known RHDs. These results strongly suggest that soil microbial community potentially harbors a wide array of organisms having diverse RHDs including those previously unknown, and that they could quickly respond to an impact of contamination of hazardous chemicals by changing the microbial community and gene diversity. PMID:19513710

Kimura, Nobutada; Kamagata, Yoichi

2009-06-10

266

Bacterial Diversity at an Acid Mine Drainage Site in Maine  

NASA Astrophysics Data System (ADS)

Bacterial diversity in acidic mine drainage at a historic Maine iron mining site was investigated by isolation of environmental DNA, PCR amplification of the V3 region of the 16S rRNA gene, denaturing gradient gel electrophoresis, and DNA sequencing.

Gaynor, J.; Sawyer, T.; Riley, F. E.; Moulton, K. D.; Rothschild, L. J.; Duboise, S. M.

2010-04-01

267

Specified Species in Gingival Crevicular Fluid Predict Bacterial Diversity  

PubMed Central

Background Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF. Methodology/Principal Findings We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms.

Asikainen, Sirkka; Dogan, Basak; Turgut, Zeynep; Paster, Bruce J.; Bodur, Aysen; Oscarsson, Jan

2010-01-01

268

Comparison of Solution Conformations and Stabilities of Modified Helix 69 rRNA Analogues from Bacteria and Human†  

PubMed Central

The helix 69 (H69) region of the large subunit (28S) rRNA of H. sapiens contains five pseudouridine (?) residues out of 19 total nucleotides, three of which are highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine in double-stranded (stem) regions. These results were compared with previous hairpin (stem plus single-stranded loop) studies in order to understand the contributions of the loop sequences to H69 structure and stability. The role of a loop nucleotide substitution from an A in bacteria (position 1918 in E. coli 23S rRNA) to a G in eukaryotes (position 3734 in H. sapiens 28S rRNA) was examined. Thermodynamic parameters for the duplex RNAs were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by circular dichroism spectroscopy. The overall folded structure of human H69 appears to be similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved; however, our results reveal subtle differences in structure and stability between the bacterial and human H69 RNAs in both the stem and loop regions. These findings may be significant with respect to H69 as a potential drug target site.

Sumita, Minako; Jiang, Jun; SantaLucia, John; Chow, Christine S.

2012-01-01

269

Short-term effects of amoxicillin on bacterial communities in manured soil.  

PubMed

Antibiotic-resistant bacteria, nutrients and antibiotics that enter the soil by means of manure may enhance the proportion of bacteria displaying antibiotic resistance among soil bacteria and may affect bacterial community structure and function. To investigate the effect of manure and amoxicillin added to manure on soil bacterial communities, microcosm experiments were performed with two soil types and the following treatments: (1) nontreated, (2) manure-treated, (3) treated with manure supplemented with 10 mg amoxicillin kg(-1) soil and (4) treated with manure supplemented with 100 mg amoxicillin kg(-1) soil, with four replicates per treatment. Manure significantly increased the total CFU count and the amoxicillin-resistant CFU count of both soil types. However, only the soil with a history of manure treatment showed a significant increase in the relative number of amoxicillin-resistant bacteria as a result of amoxicillin amendment. The majority of plasmids exogenously isolated from soil originated from soil treated with amoxicillin-supplemented manure. All 16 characterized plasmids carried the bla-TEM gene, and 10 of them belonged to the IncN group. The bla-TEM gene was detected in DNA directly extracted from soil by dot-blot hybridization of PCR amplicons and showed an increased abundance in soil samples treated with manure. Molecular fingerprint analysis of 16S rRNA gene fragments amplified from soil DNA revealed significant effects of manure and amoxicillin on the bacterial community of both soils. PMID:17991020

Binh, Chu Thi Thanh; Heuer, Holger; Gomes, Newton C Marcial; Kotzerke, Anja; Fulle, Melanie; Wilke, Bernd-Michael; Schloter, Michael; Smalla, Kornelia

2007-12-01

270

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.  

PubMed Central

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.

Rolleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

271

Bacterial meningitis  

Microsoft Academic Search

Opinion statement  Initial empiric therapy for community-acquired bacterial meningitis should be based on the possibility that penicillin-resistant\\u000a pneumococci may be the etiologic organisms and, hence, should include a combination of third-generation cephalosporin (cefotaxime\\u000a or ceftriaxone) and vancomycin. Ampicillin should be included if the patient has predisposing factors that are associated\\u000a with a risk for infection with Listeria monocytogenes. Bacterial isolates from

Karen L. Roos

1999-01-01

272

Bacterial Cystitis  

Microsoft Academic Search

Urinary tract infection is one of the most common health problems affecting patients of all ages. It is the most common nosocomial\\u000a bacterial infection in the elderly. Women are especially prone to urinary tract infections (UTIs). Although prostatitis syndrome\\u000a accounts for 25% of male office visits for genitourinary tract infections, only 5% are attributed to a bacterial cause. Acute\\u000a cystitis

Joseph B. Abdelmalak; Jeannette M. Potts

273

Non-specific biodegradation of the organophosphorus pesticides, cadusafos and ethoprophos, by two bacterial isolates.  

PubMed

An enrichment culture technique was used for the isolation of microorganisms responsible for the enhanced biodegradation of the nematicide cadusafos in soils from a potato monoculture area in Northern Greece. Mineral salts medium supplemented with nitrogen (MSMN), where cadusafos (10 mg l(-1)) was the sole carbon source, and soil extract medium (SEM) were used for the isolation of cadusafos-degrading bacteria. Two pure bacterial cultures, named CadI and CadII, were isolated and subsequently characterized by sequencing of 16S rRNA genes. Isolate CadI showed 97.4% similarity to the 16S rRNA gene of a Flavobacterium strain, unlike CadII which showed 99.7% similarity to the 16S rRNA gene of a Sphingomonas paucimobilis. Both isolates rapidly metabolized cadusafos in MSMN and SEM within 48 h with concurrent population growth. This is the first report for the isolation and characterization of soil bacteria with the ability to degrade rapidly cadusafos and use it as a carbon source. Degradation of cadusafos by both isolates was accelerated when MSMN was supplemented with glucose. In contrast, addition of succinate in MSMN marginally reduced the degradation of cadusafos. Both isolates were also able to degrade completely ethoprophos, a nematicide chemical analog of cadusafos, but did not degrade the other organophosphorus nematicides tested such as isazofos and isofenphos. Inoculation of a soil freshly treated with cadusafos or ethoprophos (10 mg l(-1)) with high inoculum densities (4.3 x 10(8) cells g(-1)) of Sphingomonas paucimobilis resulted in the rapid degradation of both nematicides. These results indicate the potential of this bacterium to be used in the clean-up of contaminated pesticide waste in the environment. PMID:16329956

Karpouzas, Dimitrios G; Fotopoulou, Anastasia; Menkissoglu-Spiroudi, Urania; Singh, Brajesh K

2005-08-01

274

The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase.  

PubMed Central

The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6

Vester, B; Hansen, L H; Douthwaite, S

1995-01-01

275

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.  

PubMed

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications. PMID:22029887

Minas, Konstantinos; McEwan, Neil R; Newbold, Charles Jamie; Scott, Karen P

2011-12-01

276

Effects of a dietary yeast extract on hematological parameters, heterophil function, and bacterial clearance in turkey poults challenged with Escherichia coli and subjected to transport stress  

Technology Transfer Automated Retrieval System (TEKTRAN)

There is a need to develop nutritional methods for controlling pathogens in poultry production. A standardized yeast extract supplement, Alphamune™ (YE), was added to turkey poult diets. Male poults were challenged by air sac injection with 60 cfu of E. coli at 1 week of age. At 3 weeks of age chal...

277

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation? †  

PubMed Central

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few ?-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.

Lauro, Federico M.; Chastain, Roger A.; Blankenship, Lesley E.; Yayanos, A. Aristides; Bartlett, Douglas H.

2007-01-01

278

Bacterial rheotaxis  

PubMed Central

The motility of organisms is often directed in response to environmental stimuli. Rheotaxis is the directed movement resulting from fluid velocity gradients, long studied in fish, aquatic invertebrates, and spermatozoa. Using carefully controlled microfluidic flows, we show that rheotaxis also occurs in bacteria. Excellent quantitative agreement between experiments with Bacillus subtilis and a mathematical model reveals that bacterial rheotaxis is a purely physical phenomenon, in contrast to fish rheotaxis but in the same way as sperm rheotaxis. This previously unrecognized bacterial taxis results from a subtle interplay between velocity gradients and the helical shape of flagella, which together generate a torque that alters a bacterium's swimming direction. Because this torque is independent of the presence of a nearby surface, bacterial rheotaxis is not limited to the immediate neighborhood of liquid–solid interfaces, but also takes place in the bulk fluid. We predict that rheotaxis occurs in a wide range of bacterial habitats, from the natural environment to the human body, and can interfere with chemotaxis, suggesting that the fitness benefit conferred by bacterial motility may be sharply reduced in some hydrodynamic conditions.

Marcos; Fu, Henry C.; Powers, Thomas R.; Stocker, Roman

2012-01-01

279

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota  

PubMed Central

Background Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p?=?0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others.

Price, Lance B.; Liu, Cindy M.; Melendez, Johan H.; Frankel, Yelena M.; Engelthaler, David; Aziz, Maliha; Bowers, Jolene; Rattray, Rogan; Ravel, Jacques; Kingsley, Chris; Keim, Paul S.; Lazarus, Gerald S.; Zenilman, Jonathan M.

2009-01-01

280

Molecular characterization of bacterial population in the forest soil of Kashmir, India  

Microsoft Academic Search

The bacterial diversity in the forest soil of Kashmir, India was investigated by 16S rDNA-dependent molecular phylogeny. Small\\u000a subunit rRNA (16S rDNA) from forest soil metagenome were amplified by polymerase chain reaction (PCR) using primers specific\\u000a to the domain bacteria. 30 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonucleases Hae 111 and Msp 1,

Nasier Ahmad; Sarojini Johri; Malik Z. Abdin; Ghulam N. Qazi

2009-01-01

281

Identification of Thermophilic Bacterial Strains Producing Thermotolerant Hydrolytic Enzymes from Manure Compost  

Microsoft Academic Search

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical\\u000a characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for

David M. Charbonneau; Fatma Meddeb-Mouelhi; Maurice Boissinot; Marc Sirois; Marc Beauregard

282

Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads.  

PubMed

Microbial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ?200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances. PMID:21460107

Bartram, Andrea K; Lynch, Michael D J; Stearns, Jennifer C; Moreno-Hagelsieb, Gabriel; Neufeld, Josh D

2011-04-01

283

A multiphasic approach for the identification of endophytic bacterial in strawberry fruit and their potential for plant growth promotion.  

PubMed

This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives showed that the isolates belonged to the species Bacillus subtilis (eight isolates), Bacillus sp. (seven isolates), Enterobacter sp. (seven isolates), Enterobacter ludwigii (six isolates), Lactobacillus plantarum (six isolates), Pseudomonas sp. (five isolates), Pantoea punctata (three isolates), and Curtobacterium citreum (three isolates). Nucleic acids were extracted from the strawberry fruit and subjected to 16S rRNA gene directed polymerase chain reaction denaturing gradient gel electrophoresis (16S rRNA PCR-DGGE). The species B. subtilis, Enterobacter sp., and Pseudomonas sp. were detected both by isolation and DGGE. The DGGE fingerprints of total bacterial DNA did not exhibit bands corresponding to several of the representative species isolated in the extinction dilution (L. plantarum, C. citreum, and P. punctata). In contrast, bands in the DGGE profile that were identified as relatives of Arthrobacter sp. and one uncultivable Erythrobacter sp. were not recovered by cultivation techniques. After isolation, the nitrogen fixation ability and the in vitro production of indole-3-acetic acid (IAA) equivalents and siderophores were evaluated. A high percentage of isolates were found to possess the ability to produce siderophores and IAA equivalents; however, only a few isolates belonging to the genera Pseudomonas and Enterobacter showed the ability to fix nitrogen. Plant growth promotion was evaluated under greenhouse conditions and revealed the ability of the Bacillus strains to enhance the number of leaves, shoot length, root dry weight, and shoot dry weight. The activity of the bacterial isolate identified as B. subtilis NA-108 exerted the greatest influence on strawberry growth and showed a 42.8% increase in number of leaves, 15.26% for high shoot, 43.5% increase in root dry weight, and a 77% increase in shoot dry weight when compared with untreated controls. PMID:21837472

de Melo Pereira, Gilberto Vinícius; Magalhães, Karina Teixeira; Lorenzetii, Emi Rainildes; Souza, Thiago Pereira; Schwan, Rosane Freitas

2011-08-12

284

Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms  

Microsoft Academic Search

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that

Sergei G. Bavykin; Yuri P. Lysov; Vladimir Zakhariev; John J. Kelly; Joany Jackman; David A. Stahl; Alexey Cherni

2004-01-01

285

Bacterial Succession in a Petroleum Land Treatment Unit  

PubMed Central

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C10 to C32 range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.

Kaplan, Christopher W.; Kitts, Christopher L.

2004-01-01

286

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis is a common cause of abnormal discharge in women of child-bearing age. It is present in 10–20% women in the UK, and may recur or regress spontaneously. It is not regarded as an STI because it can occur in virgin women, but it is more common in sexually active women. Other associations include smoking, partner change, having a

Phillip Hay

2005-01-01

287

Bacterial Biofertilizers  

Microsoft Academic Search

Many bacteria and fungi can enhance plant growth. The present review is limited to plant growth promoting rhizobacteria (PGPR). However, it includes endophytic bacteria that show plant growth enhancing activity as well. Also the best studied bacterial mechanisms of plant growth promotion are discussed, with a special emphasis on biological nitrogen fixation and synthesis of phytohormones, including less understood mechanisms

LUIS E. FUENTES-RAMIREZ; Jesus Caballero-Mellado

288

A New Approach To Utilize PCR-Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis  

Microsoft Academic Search

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5* end. The

FRANK SCHWIEGER; CHRISTOPH C. TEBBE

1998-01-01

289

Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of Bacillus and Related Genera  

Microsoft Academic Search

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-GC-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus. We found that besides the polymorphisms in the

Daniele Daffonchio; Ameur Cherif; Lorenzo Brusetti; Aurora Rizzi; Diego Mora; Abdellatif Boudabous; Sara Borin

2003-01-01

290

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota  

Microsoft Academic Search

BackgroundBacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.Methodology\\/Principal FindingsWe prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled

Lance B. Price; Cindy M. Liu; Johan H. Melendez; Yelena M. Frankel; David Engelthaler; Maliha Aziz; Jolene Bowers; Rogan Rattray; Jacques Ravel; Chris Kingsley; Paul S. Keim; Gerald S. Lazarus; Jonathan M. Zenilman; Adam J. Ratner

2009-01-01

291

Phylogenetic diversity of marine coastal picoplankton 16s rRNA genes cloned from the continental shelf off Cape Hatteras, North Carolina  

Microsoft Academic Search

The phylogenetic diversity of a continental-shelf picoplankton community was examined by analyzing 16s ribosomal RNA (rRNA) genes amplified from environmental DNA with bacterial-specific primers and the polymerase chain reaction (PCR). Picoplankton populations collected from the pycnocline (10 m) over the eastern continental shelf of the United States near Cape Hatteras, North Carolina, served as the source of bulk nucleic acids

Michael S. Rap; Paul F. Kemp; Stephen J. Giovannoni

292

Inhibitory effects of neem seed oil and its extract on various direct acting and activation-dependant mutagens-induced bacterial mutagenesis.  

PubMed

Abstract Context: Azadirachta indica A. Juss (Meliaceae), commonly called neem is a plant native to the Indian sub-continent. Neem oil extracted from the seeds of neem tree has shown promising medicinal properties. Objective: To investigate the possible anti-mutagenic activity of neem seed oil (NO) and its dimethylsulfoxide (DMSO) extract (NDE) on the mutagenicity induced by various direct acting and activation-dependant mutagens. Materials and methods: The possible anti-mutagenic activity of NO (100-10?000?µg/plate) and NDE (0.1-1000?µg/plate) as well as the mechanism of anti-mutagenic activity was studied in an in vitro Ames Salmonella/microsome assay. Results: NSO and NDE inhibited the mutagenic activity of methyl glyoxal (MG), in which case the extent of inhibition ranged from 65 to 77% and against 4-nitroquinoline-N-oxide (NQNO); it showed a 48-87% inhibition in the non-toxic doses. Similar response of NSO and NDE was seen against the activation-dependant mutagens aflatoxin B1 (AFB1, 48-88%), benzo(a)pyrene (B(a)P, 31-85%), cyclophosphamide (CP, 66-71%), 20-methylcholanthrane (20-MC, 37-83%) and acridine orange (AO, 39-72%) in the non-toxic doses. Mechanism-based studies indicated that NDE exhibits better anti-mutagenic activity in the pre- and simultaneous-treatment protocol against MG, suggesting that one or several active phytochemicals present in the extract covalently bind with the mutagen and prevent its interaction with the genome. Discussion and conclusion: These findings demonstrate that neem oil is capable of attenuating the mutagenic activity of various direct acting and activation-dependant mutagens. PMID:23998188

Vijayan, Vinod; Tiwari, Pramod Kumar; Meshram, Ghansham Pundilikji

2013-09-02

293

Terrestrial Runoff Controls the Bacterial Community Composition of Biofilms along a Water Quality Gradient in the Great Barrier Reef  

PubMed Central

16S rRNA gene molecular analysis elucidated the spatiotemporal distribution of bacterial biofilm communities along a water quality gradient. Multivariate statistics indicated that terrestrial runoff, in particular dissolved organic carbon and chlorophyll a concentrations, induced shifts of specific bacterial communities between locations and seasons, suggesting microbial biofilms could be suitable bioindicators for water quality.

Wild, Christian; Uthicke, Sven

2012-01-01

294

Control of Bacterial Motility by Environmental Factors in Polarly Flagellated and Peritrichous Bacteria Isolated from Lake Baikal  

Microsoft Academic Search

Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA

O. A. Soutourina; E. A. Semenova; V. V. Parfenova; A. Danchin; P. Bertin

2001-01-01

295

Control of rRNA transcription in Escherichia coli.  

PubMed Central

The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response.

Condon, C; Squires, C; Squires, C L

1995-01-01

296

A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance.  

PubMed

The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography--mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life. PMID:21545270

Hart, Kris M; Szpak, Michal T; Mahaney, William C; Dohm, James M; Jordan, Sean F; Frazer, Andrew R; Allen, Christopher C R; Kelleher, Brian P

2011-05-05

297

A Bacterial Enrichment Study and Overview of the Extractable Lipids from Paleosols in the Dry Valleys, Antarctica: Implications for Future Mars Reconnaissance  

NASA Astrophysics Data System (ADS)

The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography-mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life.

Hart, Kris M.; Szpak, Michal T.; Mahaney, William C.; Dohm, James M.; Jordan, Sean F.; Frazer, Andrew R.; Allen, Christopher C. R.; Kelleher, Brian P.

2011-05-01

298

Molecular diversity of drinking water bacterial communities using 16S rRNA gene sequence analyses  

EPA Science Inventory

Our understanding of the microbial community structure of drinking water distribution system has relied on culture-based methods. However, recent studies have suggested that the majority of bacteria inhabiting distribution systems are unable to grow on artificial media. The goal ...

299

Ribosomal RNA depletion for massively parallel bacterial RNA-sequencing applications.  

PubMed

RNA-sequencing (RNA-Seq) is a digital display of a transcriptome using next-generation sequencing technologies and provides detailed, high-throughput view of the transcriptome. The first step in RNA-Seq is to isolate whole transcriptome from total RNA. Since large ribosomal RNA (rRNA) constitutes approximately 90% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult using existing RNA isolation methods. RiboMinus(™) purification method provides a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large rRNA from total RNA for transcriptome analysis. It allows for whole transcriptome isolation through selective depletion of abundant rRNA molecules from total RNA. The rRNA depleted RNA fraction is termed as RiboMinus(™) RNA fraction, which is enriched in polyadenylated RNA, nonpolyadenylated RNA, preprocessed RNA, tRNA, numerous regulatory RNA molecules, and other RNA transcripts of yet unknown function. Using RiboMinus(™) method to isolate RiboMinus RNA results in up to 99.0% removal of 16S and 23S rRNA molecules from 0.5 to 10 ?g total bacterial RNA based on Bioanalyzer analysis. It enables efficient whole transcriptome sequencing analysis without major contamination from highly abundant rRNA. Residual rRNA accounts for less than 10% of entire transcriptome based on both SOLiD and Genome Analyzer RNA-Seq data. PMID:21431765

Chen, Zhoutao; Duan, Xiaoping

2011-01-01

300

Bacterial Degradation of Nicotinic Acid  

Microsoft Academic Search

A BACTERIUM utilizing nicotinic acid was isolated from culture media made up of a solution of nicotinic acid and dilute sodium sulphide and inoculated with a small amount of Potomac mud. After allowing the bacteria to grow anaerobically, several successive transfers were made into a medium containing nicotinic acid, peptone, yeast extract, metals, phosphate and sodium sulphide. The mixed bacterial

Isaac Harary

1956-01-01

301

VITCOMIC: visualization tool for taxonomic compositions of microbial communities based on 16S rRNA gene sequences  

PubMed Central

Background Understanding the community structure of microbes is typically accomplished by sequencing 16S ribosomal RNA (16S rRNA) genes. These community data can be represented by constructing a phylogenetic tree and comparing it with other samples using statistical methods. However, owing to high computational complexity, these methods are insufficient to effectively analyze the millions of sequences produced by new sequencing technologies such as pyrosequencing. Results We introduce a web tool named VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community) that can analyze millions of bacterial 16S rRNA gene sequences and calculate the overall taxonomic composition for a microbial community. The 16S rRNA gene sequences of genome-sequenced strains are used as references to identify the nearest relative of each sample sequence. With this information, VITCOMIC plots all sequences in a single figure and indicates relative evolutionary distances. Conclusions VITCOMIC yields a clear representation of the overall taxonomic composition of each sample and facilitates an intuitive understanding of differences in community structure between samples. VITCOMIC is freely available at http://mg.bio.titech.ac.jp/vitcomic/.

2010-01-01

302

New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain Chimeras†  

PubMed Central

A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

Ashelford, Kevin E.; Chuzhanova, Nadia A.; Fry, John C.; Jones, Antonia J.; Weightman, Andrew J.

2006-01-01

303

Structure of ERA in complex with the 3? end of 16S rRNA: Implications for ribosome biogenesis  

SciTech Connect

ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua; (NCI)

2009-10-09

304

Phylogenetic analysis of the bacterial communities in marine sediments.  

PubMed Central

For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed. We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments. Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis. For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed. Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes. The DNA yield was 47 micrograms per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash. Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning. Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G. J. Olsen, C. R. Woese, and R. Overbeek, J. Bacteriol. 176:1-6, 1994) with members of six major lineages represented: alpha, delta, and gamma Proteobacteria; the gram-positive high G+C content subdivision; clostridia and related organisms; and planctomyces and related organisms. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA database. The analysis of clonal representives in the first report using molecular techniques to determine the phylogenetic composition of the (eu)bacterial community present in coastal marine sediments.

Gray, J P; Herwig, R P

1996-01-01

305

Spatial distribution, diversity and composition of bacterial communities in sub-seafloor fluids at a deep-sea hydrothermal field of the Suiyo Seamount  

NASA Astrophysics Data System (ADS)

Spatial distribution, diversity, and composition of bacterial communities within the shallow sub-seafloor at the deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean, were investigated. Fluids were sampled from four boreholes in this area. Each borehole was located near or away from active vents, the distance ranging 2-40 m from active vents. In addition, fluids discharging from a natural vent and ambient seawater were sampled in this area. We extracted DNA from each sample, amplified bacterial 16S rRNA genes by PCR, cloned the PCR products and sequenced. The total number of clones analyzed was 348. Most of the detected phylotypes were affiliated with the phylum Proteobacteria, of which the detection frequency in each clone library ranged from 84.6% to 100%. The bacterial community diversity and composition were different between hydrothermal fluids and seawater, between fluids from the boreholes and the vent, and even among fluids from each borehole. The relative abundances of the phylotypes related to Thiomicrospira, Methylobacterium and Sphingomonas were significantly different among fluids from each borehole. The phylotypes related to Thiomicrospira and Alcanivorax were detected in all of the boreholes and vent samples. Our findings provide insights into bacterial communities in the shallow sub-seafloor environments at active deep-sea hydrothermal vent fields.

Kato, Shingo; Hara, Kurt; Kasai, Hiroko; Teramura, Takashi; Sunamura, Michinari; Ishibashi, Jun-ichiro; Kakegawa, Takeshi; Yamanaka, Toshiro; Kimura, Hiroyuki; Marumo, Katsumi; Urabe, Tetsuro; Yamagishi, Akihiko

2009-10-01

306

The bacterial community associated with the leech Myzobdella lugubris Leidy 1851 (Hirudinea: Piscicolidae) from Lake Erie, Michigan, USA.  

PubMed

Leeches are widespread in the Great Lakes Basin, yet their potential to harbor disease-causing agents has not been investigated. The purpose of this study was to identify the bacterial community of the commonly occurring leech, Myzobdella lugubris, within the Lake Erie Watershed. Leech samples were collected from the pectoral fins of channel catfish, Ictalurus punctatus, and freshwater drum, Aplodinotus grunniens, from Lake Erie in commercial trap nets and pooled into two samples based on host attachment. Bacteria from within the viscera of M. lugubris were identified by sequencing their 16S rRNA (rDNA) gene of amplified community bacterial DNA extracted from pooled leech homogenate samples and were checked for similarity in two public databases: the Ribosomal Database Project and BLAST. Bacteria belonging to the phylum Bacteroidetes, beta-proteobacteria, Verrucomicrobia, and unclassified Bacteria were present in the leech samples. A large number of bacteria found within leeches attached to channel catfish consisted of sequences that could not be classified beyond the Domain Bacteria. However, many of these sequences were homologous (< 45%) to the phylum Bacteroidetes. One of the five genera detected in the leech homogenates was Flavobacterium psychrophilum, a serious fish pathogen that causes Bacterial Cold Water Disease. While the occurrence of genera varies, bacteria associated with the two fish species were similar. PMID:20597437

Schulz, C; Faisal, M

2010-06-01

307

Revisiting bacterial phylogeny  

PubMed Central

Current methods used for phylogenetic classification of prokaryotes largely rely on the sequences of 16S rRNA genes that are ubiquitously present in the cell. Theoretical basis of this methodology is based on the assumption that 16S rRNA genes are only vertically inherited and are thus indigenous to each species. However, microbial genomic analysis has revealed the existence of prokaryotic species containing two types of rRNA (rrn) operons of seemingly different origins. It has also been reported that some bacteria contain 16S rRNA that are mosaics of sequences from multiple species. This suggests that horizontal gene transfer (HGT) occurred for 16S rRNA genes. In addition, a recent HGT experiment mimicking the natural HGT process has shown that a wide range of foreign 16S rRNA genes can be transferred into Escherichia coli, including those from different phylogenetic classes (with a minimum sequence identity of 80.9% to the Escherichia coli 16S rRNA gene). Thus, in contrast to the complexity hypothesis that states informational genes are rarely horizontally transferred between species, 16S rRNA is occasionally amenable to HGT. Results of the current method for rapid identification and classification of prokaryotes based on the 16S rRNA gene should thus be carefully analyzed and interpreted.

Kitahara, Kei; Miyazaki, Kentaro

2013-01-01

308

Biodegradation of Leonardite by an alkali-producing bacterial community and characterization of the degraded products.  

PubMed

In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, (13)C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer. PMID:22075634

Gao, Tong-Guo; Jiang, Feng; Yang, Jin-Shui; Li, Bao-Zhen; Yuan, Hong-Li

2011-11-11

309

Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars  

PubMed Central

Spacecraft hardware and assembly cleanroom surfaces (233 m2 in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m2) than colocated spacecraft hardware (187 OTU; 162 m2). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.

Vaishampayan, Parag; Nilsson, Henrik R.; Torok, Tamas; Venkateswaran, Kasthuri

2012-01-01

310

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars.  

PubMed

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space. PMID:22729532

La Duc, Myron T; Vaishampayan, Parag; Nilsson, Henrik R; Torok, Tamas; Venkateswaran, Kasthuri

2012-06-22

311

Field-scale transplantation experiment to investigate structures of soil bacterial communities at pioneering sites.  

PubMed

Studies on the effect of environmental conditions on plants and microorganisms are a central issue in ecology, and they require an adequate experimental setup. A strategy often applied in geobotanical studies is based on the reciprocal transplantation of plant species at different sites. We adopted a similar approach as a field-based tool to investigate the relationships of soil bacterial communities with the environment. Soil samples from two different (calcareous and siliceous) unvegetated glacier forefields were reciprocally transplanted and incubated for 15 months between 2009 and 2010. Controls containing local soils were included. The sites were characterized over time in terms of geographical (bedrock, exposition, sunlight, temperature, and precipitation) and physicochemical (texture, water content, soluble and nutrients) features. The incubating local ("home") and transplanted ("away") soils were monitored for changes in extractable nutrients and in the bacterial community structure, defined through terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA gene. Concentrations of soluble ions in most samples were more significantly affected by seasons than by the transplantation. For example, NO(3)(-) showed a seasonal pattern, increasing from 1 to 3 ?g NO(3)(-) (g soil dry weight)(-1) after the melting of snow but decreasing to <1 ?g NO(3)(-) (g soil dry weight)(-1) in autumn. Seasons, and in particular strong precipitation events occurring in the summer of 2010 (200 to 300 mm of rain monthly), were also related to changes of bacterial community structures. Our results show the suitability of this approach to compare responses of bacterial communities to different environmental conditions directly in the field. PMID:21965395

Lazzaro, Anna; Gauer, Andreas; Zeyer, Josef

2011-09-30

312

Field-Scale Transplantation Experiment To Investigate Structures of Soil Bacterial Communities at Pioneering Sites?†  

PubMed Central

Studies on the effect of environmental conditions on plants and microorganisms are a central issue in ecology, and they require an adequate experimental setup. A strategy often applied in geobotanical studies is based on the reciprocal transplantation of plant species at different sites. We adopted a similar approach as a field-based tool to investigate the relationships of soil bacterial communities with the environment. Soil samples from two different (calcareous and siliceous) unvegetated glacier forefields were reciprocally transplanted and incubated for 15 months between 2009 and 2010. Controls containing local soils were included. The sites were characterized over time in terms of geographical (bedrock, exposition, sunlight, temperature, and precipitation) and physicochemical (texture, water content, soluble and nutrients) features. The incubating local (“home”) and transplanted (“away”) soils were monitored for changes in extractable nutrients and in the bacterial community structure, defined through terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA gene. Concentrations of soluble ions in most samples were more significantly affected by seasons than by the transplantation. For example, NO3? showed a seasonal pattern, increasing from 1 to 3 ?g NO3? (g soil dry weight)?1 after the melting of snow but decreasing to <1 ?g NO3? (g soil dry weight)?1 in autumn. Seasons, and in particular strong precipitation events occurring in the summer of 2010 (200 to 300 mm of rain monthly), were also related to changes of bacterial community structures. Our results show the suitability of this approach to compare responses of bacterial communities to different environmental conditions directly in the field.

Lazzaro, Anna; Gauer, Andreas; Zeyer, Josef

2011-01-01

313

Atmospheric cloud water contains a diverse bacterial community  

NASA Astrophysics Data System (ADS)

Atmospheric cloud water contains an active microbial community which can impact climate, human health and ecosystem processes in terrestrial and aquatic systems. Most studies on the composition of microbial communities in clouds have been performed with orographic clouds that are typically in direct contact with the ground. We collected water samples from cumulus clouds above the upper U.S. Midwest. The cloud water was analyzed for the diversity of bacterial phylotypes by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene amplicons. DGGE analyses of bacterial communities detected 17-21 bands per sample. Sequencing confirmed the presence of a diverse bacterial community; sequences from seven bacterial phyla were retrieved. Cloud water bacterial communities appeared to be dominated by members of the cyanobacteria, proteobacteria, actinobacteria and firmicutes.

Kourtev, Peter S.; Hill, Kimberly A.; Shepson, Paul B.; Konopka, Allan

2011-09-01

314

Bacterial Rheotaxis  

NASA Astrophysics Data System (ADS)

Rheotaxis is the directed movement of an organism resulting from fluid velocity gradients, long studied in fish, aquatic invertebrates and spermatozoa. Here we show that rheotaxis also occurs in bacteria. Using controlled microfluidic shear flows, we demonstrate and quantify rheotaxis in Bacillus subtilis. A mathematical model of a bacterium swimming in a shear flow is in good agreement with observations and reveals that bacterial rheotaxis results from a subtle interplay between velocity gradients and the helical shape of flagella, which together generate a torque that reorients the cell, altering its swimming direction. The magnitude of the observed rheotactic velocity is comparable to typical chemotactic velocities, suggesting that rheotaxis can interfere with bacterial processes based on directed motility, such as foraging and infection.

Marcos, Marcos; Fu, Henry; Powers, Thomas; Stocker, Roman

2011-11-01

315

Cryptomonad nuclear and nucleomorph 18S rRNA phylogeny  

Microsoft Academic Search

Nuclear and nucleomorph 18S ribosomal RNA genes from six cryptomonads were amplified by the polymerase chain reaction and sequenced. Phylogenetic trees were constructed by distance, parsimony, and maximum likelihood methods for all available cryptomonad nuclear and nucleomorph 18S rRNA sequences. Nuclear and nucleomorph trees are largely congruent and clearly disprove the idea of polyphyletic origins for cryptomonad chloroplasts. Both show

T. Cavalier-Smith; J. A. Couch; K. E. Thorsteinsen; P. Gilson; J. A. Deane; D. R. A. Hill; G. I. Mcfadden

1996-01-01

316

Deoxygenation alters bacterial diversity and community composition in the ocean's largest oxygen minimum zone.  

PubMed

Oceanic oxygen minimum zones (OMZs) have a central role in biogeochemical cycles and are expanding as a consequence of climate change, yet how deoxygenation will affect the microbial communities that control these cycles is unclear. Here we sample across dissolved oxygen gradients in the oceans' largest OMZ and show that bacterial richness displays a unimodal pattern with decreasing dissolved oxygen, reaching maximum values on the edge of the OMZ and decreasing within it. Rare groups on the OMZ margin are abundant at lower dissolved oxygen concentrations, including sulphur-cycling Chromatiales, for which 16S rRNA was amplified from extracted RNA. Microbial species distribution models accurately replicate community patterns based on multivariate environmental data, demonstrate likely changes in distributions and diversity in the eastern tropical North Pacific Ocean, and highlight the sensitivity of key bacterial groups to deoxygenation. Through these mechanisms, OMZ expansion may alter microbial composition, competition, diversity and function, all of which have implications for biogeochemical cycling in OMZs. PMID:24162368

Beman, J Michael; Carolan, Molly T

2013-10-28

317

Wide Distribution and Diversity of Members of the Bacterial Kingdom Acidobacterium in the Environment  

PubMed Central

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.

Barns, Susan M.; Takala, Shannon L.; Kuske, Cheryl R.

1999-01-01

318

Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.  

PubMed

In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66?% of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52?% of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90?% of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method. PMID:22837218

Meex, Cécile; Neuville, Florence; Descy, Julie; Huynen, Pascale; Hayette, Marie-Pierre; De Mol, Patrick; Melin, Pierrette

2012-07-26

319

pH dynamics and bacterial community composition in the rumen of lactating dairy cows.  

PubMed

The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH=6.11 to 6.51, diurnal pH range=0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean= <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii. PMID:20059926

Palmonari, A; Stevenson, D M; Mertens, D R; Cruywagen, C W; Weimer, P J

2010-01-01

320

Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies  

PubMed Central

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m7G1405) or N1-methyl adenosine 1408 (m1A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in E. coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-L-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.

Zelinskaya, Natalia; Rankin, C. Robert; Macmaster, Rachel; Savic, Miloje; Conn, Graeme L.

2010-01-01

321

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis.  

PubMed

The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency. PMID:23970567

Yano, Koichi; Wada, Tetsuya; Suzuki, Shota; Tagami, Kazumi; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Kawaguchi, Yasuhiro; Masuda, Kenta; Akanuma, Genki; Nanamiya, Hideaki; Niki, Hironori; Yoshikawa, Hirofumi; Kawamura, Fujio

2013-08-22

322

Characterization of Bacterial Community Structure on a Weathered Pegmatitic Granite  

Microsoft Academic Search

This study exploited the contrasting major element chemistry of a pegmatitic granite to investigate mineralogical influences on bacterial community structure. Intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were extracted, together with whole-rock granite. Environmental scanning electron microscopy revealed a diversity of bacterial structures, with rods and cocci clearly visible on surfaces of all mineral types. Bacterial automated

Deirdre B. Gleeson; Nabla M. Kennedy; Nicholas Clipson; Karrie Melville; Geoffrey M. Gadd; Frank P. McDermott

2006-01-01

323

Diversity of iron oxidizers in wetland soils revealed by novel 16S rRNA primers targeting Gallionella-related bacteria.  

PubMed

Neutrophilic iron-oxidizing bacteria (FeOB) are important catalysts of iron cycling in wetland environments. However, little is known about their diversity and distribution in various environments. The aim of this study was to develop a PCR-DGGE assay enabling the detection of neutrophilic iron oxidizers in wetland habitats. Gradient tubes were used to enrich FeOB. From these enrichments, a clone library was established on the basis of the almost complete 16S rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly alpha- and beta-Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, followed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella sp. On the basis of their nucleotide sequences, four groups could be identified that were comparable to the DGGE banding pattern obtained before with the same PCR products as used for the second clone library. Using these Gallionella-specific 16S rRNA gene-based primers, in combination with DGGE, first insights into the diversity and distribution of these bacteria in wetland soils were obtained. PMID:19225553

Wang, Juanjuan; Muyzer, Gerard; Bodelier, Paul L E; Laanbroek, Hendrikus J

2009-02-19

324

Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.  

PubMed Central

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence. Images

Rehnstam, A S; Norqvist, A; Wolf-Watz, H; Hagstrom, A

1989-01-01

325

Serenoa repens associated with Urtica dioica (ProstaMEV) and curcumin and quercitin (FlogMEV) extracts are able to improve the efficacy of prulifloxacin in bacterial prostatitis patients: results from a prospective randomised study.  

PubMed

We report the results of a prospective randomised study to evaluate the therapeutic effect of Serenoa repens, Urtica dioica (ProstaMEV), quercitin and curcumin (FlogMEV) extracts associated with prulifloxacin in patients affected by chronic bacterial prostatitis (CBP). From a whole population of 284 patients, 143 patients affected by CBP [National Institutes of Health (NIH) class II prostatitis] were enrolled. All patients received prulifloxacin 600 mg daily for 14 days, in accordance with antibiogram results. Patients were split into two groups: Group A received prulifloxacin associated with ProstaMEV and FlogMEV; Group B received only antibiotic therapy. Microbiological and clinical efficacies were tested by two follow-up visits at 1 month and 6 months, respectively. Quality of life (QoL) was measured using the NIH Chronic Prostatitis Symptom Index (CPSI) and International Prostatic Symptom Score (IPSS) questionnaires. Group A comprised 106 patients and Group B comprised 37 patients. One month after treatment, 89.6% of patients who had received prulifloxacin associated with ProstaMEV and FlogMEV did not report any symptoms related to CBP, whilst only 27% of patients who received antibiotic therapy alone were recurrence-free (P < 0.0001). Significant differences were found between groups in terms of symptoms and QoL (P < 0.0001 for both). Six months after treatment, no patients in Group A had recurrence of disease whilst two patients in Group B did. Questionnaire results demonstrated statistically significant differences between groups (all P < 0.001). The association of S. repens, U. dioica (ProstaMEV), quercitin and curcumin (FlogMEV) extracts is able to improve the clinical efficacy of prulifloxacin in patients affected by CBP. PMID:19181486

Cai, Tommaso; Mazzoli, Sandra; Bechi, Adriano; Addonisio, Patrizia; Mondaini, Nicola; Pagliai, Roberto Castricchi; Bartoletti, Riccardo

2009-01-31

326

Bacterial Growth  

NSDL National Science Digital Library

Dr. Brett Finlay shows how bacteria can grow rapidly to incredible numbers, and also explains what limits this explosive growth. This resource would be great preparation material for a classroom discussion or video presentation for both the students and the teacher. This visual helps further broaden the knowledge of students in both the upper high school and college undergraduate on bacterial growth. The lecture is featured on the DVD 2000 and Beyond: Confronting the Microbe Menace, available free from HHMI. The video is 54 seconds long and available on WMV (10MB) and MOV (8MB). All Infection Disease videos can be found at http://www.hhmi.org/biointeractive/disease/video.html .

Howard Hughes Medical Institute (;)

2007-03-27

327

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.  

PubMed Central

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.

Lee, D H; Zo, Y G; Kim, S J

1996-01-01

328

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis  

PubMed Central

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

Layton, A. C.; Karanth, P. N.; Lajoie, C. A.; Meyers, A. J.; Gregory, I. R.; Stapleton, R. D.; Taylor, D. E.; Sayler, G. S.

2000-01-01

329

Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles  

PubMed Central

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.

Woo, Patrick C. Y.; Ng, Kenneth H. L.; Lau, Susanna K. P.; Yip, Kam-tong; Fung, Ami M. Y.; Leung, Kit-wah; Tam, Dorothy M. W.; Que, Tak-lun; Yuen, Kwok-yung

2003-01-01

330

Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting.  

PubMed

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI-TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood-borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI-TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI-TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains. PMID:23334977

Böhme, Karola; Fernández-No, Inmaculada C; Pazos, Manuel; Gallardo, José M; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2013-02-25

331

Empirical and Theoretical Bacterial Diversity in Four Arizona Soils  

Microsoft Academic Search

Understanding patterns of biodiversity in microbial communities is severely constrained by the difficulty of adequately sampling these complex systems. We illustrate the problem with empirical data from small surveys (200-member 16S rRNA gene clone libraries) of four bacterial soil communities from two locations in Arizona. Among the four surveys, nearly 500 species-level groups (Dunbar et al., Appl. Environ. Microbiol. 65:662-1669,

John Dunbar; Susan M. Barns; Lawrence O. Ticknor; Cheryl R. Kuske

2002-01-01

332

Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring  

Microsoft Academic Search

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA

PHILIP HUGENHOLTZ; CHRISTIAN PITULLE; KAREN L. HERSHBERGER; NORMAN R. PACE

1998-01-01

333

Polyadenylation of rRNA in Saccharomyces cerevisiae.  

PubMed

In contrast to mRNAs, rRNAs are transcribed by RNA polymerase I or III and are not believed to be polyadenylated. Here we show that in Saccharomyces cerevisiae, at least a small fraction of rRNAs do have a poly(A) tail. The levels of polyadenylated rRNAs are dramatically increased in strains lacking the degradation function of Rrp6p, a component of the nuclear exosome. Pap1p, the poly(A) polymerase, is responsible for adenylating the rRNAs despite the fact that the rRNAs do not have a canonical polyadenylation signal. Polyadenylated rRNAs reside mainly within the nucleus and are in turn degraded. For at least one rRNA type, the polyadenylation preferentially occurs on the precursor rather than the mature product. The existence of polyadenylated rRNAs may reflect a quality-control mechanism of rRNA biogenesis. PMID:15173578

Kuai, Letian; Fang, Feng; Butler, J Scott; Sherman, Fred

2004-06-01

334

An Intergenic Non-Coding rRNA Correlated with Expression of the rRNA and Frequency of an rRNA Single Nucleotide Polymorphism in Lung Cancer Cells  

PubMed Central

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

Gu, Yuhan D.; Kasprzak, Wojciech; Hwang, Christopher J.; Fields, Janet R.; Leighty, Robert M.; Quinones, Octavio; Shapiro, Bruce A.; Alvord, W. Gregory; Anderson, Lucy M.

2009-01-01

335

16S rRNA oligonucleotide catalog data base.  

PubMed Central

We have developed a package of programs to create, maintain and manipulate a data base of 16S rRNA oligonucleotide catalog data. For the first time all the published catalog data is brought together in one place in a readily usable form. The package allows generation of dendrograms, facilitates searches for related oligonucleotides between catalogs, and allows construction of global and local dictionaries. Interactive capabilities allow for searches of the dictionary as well as an associated file of likely oligonucleotide families.

Sobieski, J M; Chen, K N; Filiatreau, J C; Pickett, M H; Fox, G E

1984-01-01

336

The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species.  

PubMed Central

Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies. Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R. marinus diverges sharply from major bacterial phyla and is most closely allied to the Flexibacter-Cytophaga-Bacteroides group. Further analyses revealed that the R. marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNA(Ile) and tRNA(Ala). Images

Andresson, O S; Fridjonsson, O H

1994-01-01

337

Robust Computational Analysis of rRNA Hypervariable Tag Datasets  

PubMed Central

Next-generation DNA sequencing is increasingly being utilized to probe microbial communities, such as gastrointestinal microbiomes, where it is important to be able to quantify measures of abundance and diversity. The fragmented nature of the 16S rRNA datasets obtained, coupled with their unprecedented size, has led to the recognition that the results of such analyses are potentially contaminated by a variety of artifacts, both experimental and computational. Here we quantify how multiple alignment and clustering errors contribute to overestimates of abundance and diversity, reflected by incorrect OTU assignment, corrupted phylogenies, inaccurate species diversity estimators, and rank abundance distribution functions. We show that straightforward procedural optimizations, combining preexisting tools, are effective in handling large () 16S rRNA datasets, and we describe metrics to measure the effectiveness and quality of the estimators obtained. We introduce two metrics to ascertain the quality of clustering of pyrosequenced rRNA data, and show that complete linkage clustering greatly outperforms other widely used methods.

Sipos, Maksim; Jeraldo, Patricio; Chia, Nicholas; Qu, Ani; Dhillon, A. Singh; Konkel, Michael E.; Nelson, Karen E.; White, Bryan A.; Goldenfeld, Nigel

2010-01-01

338

Cladistic analysis of 5S rRNA and 16S rRNA secondary and primary structure—The evolution of eukaryotes and their relation to archaebacteria  

Microsoft Academic Search

Summary The secondary structure of 5S rRNA has been elucidated by a cladistic analysis resulting in minimal models for eukaryotes, eubacteria, and halophilic-methanogenic archaebacteria, as well as for an ur-5S rRNA. This ancestor of all present-day 5S rRNA molecules is compared with an ur-tRNA and can be fitted into a tRNA-like structure allowing tertiary-structure interactions at the equivalent positions. A

Jörn Wolters; Volker A. Erdmann

1986-01-01

339

Early succession of bacterial biofilms in paper machines.  

PubMed

Formation of biofilms causes severe problems in paper machines, and hence financial costs. It would be preferable to prevent attachment of the primary-colonizing bacteria than to control the growth of secondary communities, which are sheltered by exopolysaccharide slime layers. We have therefore investigated the early succession of paper-machine biofilms by incubating stainless-steel test coupons in the process water-flow lines in two paper machines operating in slightly alkaline conditions in temperatures (45 and 49 degrees C) supporting thermophilic microbes. Microbial succession was profiled using length heterogeneity analysis of PCR-amplified 16S rRNA genes (LH-PCR) and linking the sequence data of the created 16S rRNA gene libraries to the dominant LH-PCR peaks. Although the bacterial fingerprints obtained from the attached surface communities varied slightly in different samples, the biomarker signals of the dominating primary-colonizing bacterial groups remained high over time in each paper machine. Most of the 16S rRNA gene copies in the early biofilms were assigned to the genera Rhodobacter, Tepidimonas, and Cloacibacterium. The dominance of these sequence types decreased in the developing biofilms. Finally, as phylogenetically identical primary-colonizers were detected in the two different paper mills, the machines evidently had similar environmental conditions for bacterial growth and potentially a common source of contamination. PMID:19390885

Tiirola, Marja; Lahtinen, Tomi; Vuento, Matti; Oker-Blom, Christian

2009-04-24

340

Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance?  

PubMed Central

The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid. Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C2055A-A2572U had remarkable synergistic effects on linezolid resistance relative to the effects of the corresponding single mutations. This study emphasizes that effects of rRNA mutations at the PTC are organism dependent. Moreover, the data show a nonpredictable cross-resistance pattern between linezolid, chloramphenicol, clindamycin, and valnemulin. The data underscore the significance of mutations at distal nucleotides, either alone or in combination with other mutated nucleotides, in contributing to linezolid resistance.

Long, Katherine S.; Munck, Christian; Andersen, Theis M. B.; Schaub, Maria A.; Hobbie, Sven N.; Bottger, Erik C.; Vester, Birte

2010-01-01

341

The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies.  

PubMed

Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment. PMID:23977026

Ghyselinck, Jonas; Pfeiffer, Stefan; Heylen, Kim; Sessitsch, Angela; De Vos, Paul

2013-08-19

342

Bacterial Community Structure and Diversity in a Century-Old Manure-Treated Agroecosystem  

Microsoft Academic Search

Changes in soil microbial community structure and diversity may reflect environmental impact. We exam- ined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by

H. Y. Sun; S. P. Deng; W. R. Raun

2004-01-01

343

Seasonal dynamics of the bacterial community in Lake Namco, the largest Tibetan lake  

Microsoft Academic Search

Seasonal variations of bacterial abundance and diversity in Lake Namco, the large and deep high altitude lake, were first investigated using flow cytometry enumeration and 16S rRNA gene clone library analysis. Bacterial abundance varied from 1.5×10 to 12.3×10 cells mL, and exhibited a seasonal pattern that correlated with water temperature and phytoplankton abundance rather than nutrient. This indicated that temperature

Yongqin Liu; Tandong Yao; Nianzhi Jiao; Xiaobo Liu; Shichang Kang; Tingwei Luo

2012-01-01

344

Bacterial Community of the Largest Oligosaline Lake, Namco on the Tibetan Plateau  

Microsoft Academic Search

Bacterial abundances and diversity in the surface water of Lake Namco, the largest oligosaline lake on the Tibetan Plateau, were examined using flow cytometry approach and constructing 16S rRNA gene clone libraries. Bacterial abundances were from 0.08 × 10 to 1.6 × 10 cells mL, and were in the reported range of other lakes of the Tibetan Plateau and high

Xiaobo Liu; Tandong Yao; Shichang Kang; Nianzhi Jiao; Yonghui Zeng; Yongqin Liu

2010-01-01

345

Characterization of a Bacterial Community in an Abandoned Semiarid Lead-Zinc Mine Tailing Site  

Microsoft Academic Search

Bacterial diversity in mine tailing microbial communities has not been thoroughly investigated despite the correlations that have been observed between the relative microbial diversity and the success of revegetation efforts at tailing sites. This study employed phylogenetic analyses of 16S rRNA genes to compare the bacterial communities present in highly disturbed, extremely (pH 2.7) and moderately (pH 5.7) acidic lead-zinc

Monica O. Mendez; Julia W. Neilson; Raina M. Maier

2008-01-01

346

Partitioning of bacterial communities between seawater and healthy, black band diseased, and dead coral surfaces.  

PubMed

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue. PMID:11976091

Frias-Lopez, Jorge; Zerkle, Aubrey L; Bonheyo, George T; Fouke, Bruce W

2002-05-01

347

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture  

PubMed Central

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones analysed. In all, 28 phylotypes were identified: Lysobacter enzymogenes was the most abundant phylotype (31.4%), followed by Lysobacter sp. C3 (28.3%), gamma proteobacterium N4-7 (6.6%), Methylobacterium SM4 (4.7%) and Staphylococcus epidermidis (4.7%); 36 clones (7.0%) represented uncultivable phylotypes. We conclude that a diverse range of bacterial species are found within biofilms on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties.

Dempsey, Kate E; Riggio, Marcello P; Lennon, Alan; Hannah, Victoria E; Ramage, Gordon; Allan, David; Bagg, Jeremy

2007-01-01

348

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture.  

PubMed

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones analysed. In all, 28 phylotypes were identified: Lysobacter enzymogenes was the most abundant phylotype (31.4%), followed by Lysobacter sp. C3 (28.3%), gamma proteobacterium N4-7 (6.6%), Methylobacterium SM4 (4.7%) and Staphylococcus epidermidis (4.7%); 36 clones (7.0%) represented uncultivable phylotypes. We conclude that a diverse range of bacterial species are found within biofilms on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties. PMID:17501992

Dempsey, Kate E; Riggio, Marcello P; Lennon, Alan; Hannah, Victoria E; Ramage, Gordon; Allan, David; Bagg, Jeremy

2007-01-01

349

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.  

PubMed Central

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.

Zheng, D; Alm, E W; Stahl, D A; Raskin, L

1996-01-01

350

Antarctic ice core samples: culturable bacterial diversity.  

PubMed

Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 × 10(3) CFU ml(-1) of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2-100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 °C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. PMID:23041141

Shivaji, Sisinthy; Begum, Zareena; Shiva Nageswara Rao, Singireesu Soma; Vishnu Vardhan Reddy, Puram V; Manasa, Poorna; Sailaja, Buddi; Prathiba, Mambatta S; Thamban, Meloth; Krishnan, Kottekkatu P; Singh, Shiv M; Srinivas, Tanuku N R

2012-10-04

351

Phylogenetic analysis of oryx species using partial sequences of mitochondrial rRNA genes.  

PubMed

We conducted a comparative evaluation of 12S rRNA and 16S rRNA genes of the mitochondrial genome for molecular differentiation among three oryx species (Oryx leucoryx, Oryx dammah and Oryx gazella) with respect to two closely related outgroups, addax and roan. Our findings showed the failure of 12S rRNA gene to differentiate between the genus Oryx and addax, whereas a 342-bp partial sequence of 16S rRNA accurately grouped all five taxa studied, suggesting the utility of 16S rRNA segment for molecular phylogeny of oryx at the genus and possibly species levels. PMID:19048493

Khan, H A; Arif, I A; Al Farhan, A H; Al Homaidan, A A

2008-10-28

352

Interactions between bacterial populations during degradation of a lubricant base oil  

Microsoft Academic Search

The composition of microbial communities degrading trimethylolpropaneoleate, a synthetic fatty acid ester, was studied under conditions similar to a frequently used degradation test. A combination of conventional phenotypic tests and molecular biological methods, internal rRNA gene spacer amplification and randomly amplified DNA assay, was used for strain monitoring. Few bacterial species were selected from the large diversity of the inocula

Silke Sonderkamp; Anja Vomberg; Christoph Schmitz; Ulrich Faßbender; Ulrich Klinner

2001-01-01

353

Flow cytometric determination of bacterial populations in bottled natural mineral waters  

Microsoft Academic Search

In order to enhance the quality and safety of bottled natural mineral waters, new methodologies besides classical bacteriology have been evaluated. Multi laser flow cytometry has been used to identify bacterial populations based on their DNA content, physiological activity and phylogeny from in situ hybridization with rRNA targeted DNA probes. Due to the low content of organic material in these

Wolfgang Beisker; H. Meier

1998-01-01

354

Growth rate regulation of rRNA content of a marine Synechococcus (cyanobacterium) strain  

SciTech Connect

The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells. The relationship between growth rate and cellular rRNA content comprised three phases: (1) at low growth rates, rRNA cell{sup {minus}1} remained approximately constant; (2) at intermediate rates, rRNA cell{sup {minus}1} increased proportionally with growth rate; and (3) at the highest, light-saturated rates, rRNA cell{sup {minus}1} dropped abruptly. Total cellular RNA was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration were related to growth rate in a manner similar to rRNA cell{sup {minus}1}, although the overall magnitude linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.

Binder, B.J.; Liu, Y.C. [Univ. of Georgia, Athens, GA (United States). Dept. of Marine Sciences

1998-09-01

355

Bacterial penetration of restored cavities.  

PubMed

The aim of this study was to assess the quality of the marginal seals of 7 restoratives by means of a bacterial penetration test in vitro. Sixty intact premolars and third molars that were scheduled for extraction were used in the test. There were 2 experimental groups of teeth, as follows: (1) A class V conventional cavity and a wedge erosion cavity were prepared on the buccal surface and the lingual surface, respectively, of each tooth. (2) A class V conventional cavity and a wedge erosion cavity were prepared on the buccal surface and the lingual surface, respectively, of each tooth with a completely removed enamel layer. The cavities were then reconstructed with different restorative materials. The quality of the marginal seals was evaluated by submerging the teeth in a bacterial suspension and incubating them in an anaerobic milieu at 37 degrees C for 20 hours. The teeth were subsequently processed for histologic data and bacterial staining. The best marginal sealing in both the wedge erosion and the class V cavities was provided by the Herculite/Optibond system and the Valux Plus/Scotchbond Multipurpose system. Bacterial penetration was slightly greater with the Luxat compomer and the Dyrect compomer, as well as with Vitremer glass ionomer cement and Fuji LC glass ionomer cement. The bacterial penetration test showed that the use of restorative material does not entirely eliminate microleakage. PMID:11250635

Zivkovi?, S; Bojovi?, S; Pavlica, D

2001-03-01

356

New Treatments for Bacterial Keratitis  

PubMed Central

Purpose. To review the newer treatments for bacterial keratitis. Data Sources. PubMed literature search up to April 2012. Study Selection. Key words used for literature search: “infectious keratitis”, “microbial keratitis”, “infective keratitis”, “new treatments for infectious keratitis”, “fourth generation fluoroquinolones”, “moxifloxacin”, “gatifloxacin”, “collagen cross-linking”, and “photodynamic therapy”. Data Extraction. Over 2400 articles were retrieved. Large scale studies or publications at more recent dates were selected. Data Synthesis. Broad spectrum antibiotics have been the main stay of treatment for bacterial keratitis but with the emergence of bacterial resistance; there is a need for newer antimicrobial agents and treatment methods. Fourth-generation fluoroquinolones and corneal collagen cross-linking are amongst the new treatments. In vitro studies and prospective clinical trials have shown that fourth-generation fluoroquinolones are better than the older generation fluoroquinolones and are as potent as combined fortified antibiotics against common pathogens that cause bacterial keratitis. Collagen cross-linking was shown to improve healing of infectious corneal ulcer in treatment-resistant cases or as an adjunct to antibiotics treatment. Conclusion. Fourth-generation fluoroquinolones are good alternatives to standard treatment of bacterial keratitis using combined fortified topical antibiotics. Collagen cross-linking may be considered in treatment-resistant infectious keratitis or as an adjunct to antibiotics therapy.

Wong, Raymond L. M.; Gangwani, R. A.; Yu, Lester W. H.; Lai, Jimmy S. M.

2012-01-01

357

Response and resilience of soil biocrust bacterial communities to chronic physical disturbance in arid shrublands  

PubMed Central

The impact of 10 years of annual foot trampling on soil biocrusts was examined in replicated field experiments at three cold desert sites of the Colorado Plateau, USA. Trampling detrimentally impacted lichens and mosses, and the keystone cyanobacterium, Microcoleus vaginatus, resulting in increased soil erosion and reduced C and N concentrations in surface soils. Trampled biocrusts contained approximately half as much extractable DNA and 20–52% less chlorophyll a when compared with intact biocrusts at each site. Two of the three sites also showed a decline in scytonemin-containing, diazotrophic cyanobacteria in trampled biocrusts. 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses of soil bacteria from untrampled and trampled biocrusts demonstrated a reduced proportion (23–65% reduction) of M. vaginatus and other Cyanobacteria in trampled plots. In parallel, other soil bacterial species that are natural residents of biocrusts, specifically members of the Actinobacteria, Chloroflexi and Bacteroidetes, became more readily detected in trampled than in untrampled biocrusts. Replicate 16S rRNA T-RFLP profiles from trampled biocrusts at all three sites contained significantly more fragments (n=17) than those of untrampled biocrusts (n?6) and exhibited much higher variability among field replicates, indicating transition to an unstable disturbed state. Despite the dramatic negative impacts of trampling on biocrust physical structure and composition, M. vaginatus could still be detected in surface soils after 10 years of annual trampling, suggesting the potential for biocrust re-formation over time. Physical damage of biocrusts, in concert with changing temperature and precipitation patterns, has potential to alter performance of dryland ecosystems for decades.

Kuske, Cheryl R; Yeager, Chris M; Johnson, Shannon; Ticknor, Lawrence O; Belnap, Jayne

2012-01-01

358

Response and resilience of soil biocrust bacterial communities to chronic physical disturbance in arid shrublands.  

PubMed

The impact of 10 years of annual foot trampling on soil biocrusts was examined in replicated field experiments at three cold desert sites of the Colorado Plateau, USA. Trampling detrimentally impacted lichens and mosses, and the keystone cyanobacterium, Microcoleus vaginatus, resulting in increased soil erosion and reduced C and N concentrations in surface soils. Trampled biocrusts contained approximately half as much extractable DNA and 20-52% less chlorophyll a when compared with intact biocrusts at each site. Two of the three sites also showed a decline in scytonemin-containing, diazotrophic cyanobacteria in trampled biocrusts. 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses of soil bacteria from untrampled and trampled biocrusts demonstrated a reduced proportion (23-65% reduction) of M. vaginatus and other Cyanobacteria in trampled plots. In parallel, other soil bacterial species that are natural residents of biocrusts, specifically members of the Actinobacteria, Chloroflexi and Bacteroidetes, became more readily detected in trampled than in untrampled biocrusts. Replicate 16S rRNA T-RFLP profiles from trampled biocrusts at all three sites contained significantly more fragments (n = 17) than those of untrampled biocrusts (n?6) and exhibited much higher variability among field replicates, indicating transition to an unstable disturbed state. Despite the dramatic negative impacts of trampling on biocrust physical structure and composition, M. vaginatus could still be detected in surface soils after 10 years of annual trampling, suggesting the potential for biocrust re-formation over time. Physical damage of biocrusts, in concert with changing temperature and precipitation patterns, has potential to alter performance of dryland ecosystems for decades. PMID:22113374

Kuske, Cheryl R; Yeager, Chris M; Johnson, Shannon; Ticknor, Lawrence O; Belnap, Jayne

2011-11-24

359

Bacterial diversity within the human subgingival crevice  

PubMed Central

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

Kroes, Ian; Lepp, Paul W.; Relman, David A.

1999-01-01

360

Bacterial Community Reconstruction Using Compressed Sensing  

NASA Astrophysics Data System (ADS)

Bacteria are the unseen majority on our planet, with millions of species and comprising most of the living protoplasm. We propose a novel approach for reconstruction of the composition of an unknown mixture of bacteria using a single Sanger-sequencing reaction of the mixture. Our method is based on compressive sensing theory, which deals with reconstruction of a sparse signal using a small number of measurements. Utilizing the fact that in many cases each bacterial community is comprised of a small subset of all known bacterial species, we show the feasibility of this approach for determining the composition of a bacterial mixture. Using simulations, we show that sequencing a few hundred base-pairs of the 16S rRNA gene sequence may provide enough information for reconstruction of mixtures containing tens of species, out of tens of thousands, even in the presence of realistic measurement noise. Finally, we show initial promising results when applying our method for the reconstruction of a toy experimental mixture with five species. Our approach may have a potential for a simple and efficient way for identifying bacterial species compositions in biological samples.

Amir, Amnon; Zuk, Or

361

Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis  

PubMed Central

Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health.

Gross, Erin L.; Beall, Clifford J.; Kutsch, Stacey R.; Firestone, Noah D.; Leys, Eugene J.; Griffen, Ann L.

2012-01-01

362

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.  

PubMed Central

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

Liu, W T; Marsh, T L; Cheng, H; Forney, L J

1997-01-01

363

Bacterial biodiversity from anthropogenic extreme environments: a hyper-alkaline and hyper-saline industrial residue contaminated by chromium and iron.  

PubMed

Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg(-1)) and iron (from 2 to 108 g kg(-1)). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to Bacillus and Lysobacter genera. Lysobacter related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to Thiobacillus were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with Bacillus (already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest. PMID:22350256

Brito, Elcia M S; Piñón-Castillo, Hilda A; Guyoneaud, Rémy; Caretta, César A; Gutiérrez-Corona, J Félix; Duran, Robert; Reyna-López, Georgina E; Nevárez-Moorillón, G Virginia; Fahy, Anne; Goñi-Urriza, Marisol

2012-02-17

364

Sputum Microbiota in Tuberculosis as Revealed by 16S rRNA Pyrosequencing  

PubMed Central

Background Tuberculosis (TB) remains a global threat in the 21st century. Traditional studies of the disease are focused on the single pathogen Mycobacterium tuberculosis. Recent studies have revealed associations of some diseases with an imbalance in the microbial community. Characterization of the TB microbiota could allow a better understanding of the disease. Methodology/Principal Findings Here, the sputum microbiota in TB infection was examined by using 16S rRNA pyrosequencing. A total of 829,873 high-quality sequencing reads were generated from 22 TB and 14 control sputum samples. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were the five major bacterial phyla recovered, which together composed over 98% of the microbial community. Proteobacteria and Bacteroidetes were more represented in the TB samples and Firmicutes was more predominant in the controls. Sixteen major bacterial genera were recovered. Streptococcus, Neisseria and Prevotella were the most predominant genera, which were dominated by several operational taxonomic units grouped at a 97% similarity level. Actinomyces, Fusobacterium, Leptotrichia, Prevotella, Streptococcus, and Veillonella were found in all TB samples, possibly representing the core genera in TB sputum microbiota. The less represented genera Mogibacterium, Moryella and Oribacterium were enriched statistically in the TB samples, while a genus belonging to the unclassified Lactobacillales was enriched in the controls. The diversity of microbiota was similar in the TB and control samples. Conclusions/Significance The composition and diversity of sputum microbiota in TB infection was characterized for the first time by using high-throughput pyrosequencing. It lays the framework for examination of potential roles played by the diverse microbiota in TB pathogenesis and progression, and could ultimately facilitate advances in TB treatment.

Law, Patrick Tik Wan; Au, Chun Hang; Nong, Wenyan; Kam, Kai Man; Kwan, Hoi Shan; Tsui, Stephen Kwok Wing

2013-01-01

365

YhiQ is RsmJ, the methyltransferase responsible for methylation of G1516 in 16S rRNA of E. coli.  

PubMed

Ten methyltransferases and one pseudouridine synthase are required for complete modification of the small ribosomal subunit in Escherichia coli. Nine methyltransferases, as well as the pseudouridine synthase, are already known. Here, we identify RsmJ, the last unknown methyltransferase required for methylation of m(2)G1516 in 16S ribosomal RNA (rRNA), as the protein encoded by yhiQ. Reverse transcription primer extension analysis reveals that rRNA extracted from a yhiQ deletion strain is not methylated at G1516. Moreover, methylation is restored upon gene complementation. Also, purified recombinant YhiQ specifically methylates 30S subunits extracted from the deletion strain. The absence of the yhiQ gene leads to a cold-sensitive phenotype. Based on these data, we propose that the yhiQ gene be renamed rsmJ. PMID:22079366

Basturea, Georgeta N; Dague, Darryl R; Deutscher, Murray P; Rudd, Kenneth E

2011-11-04

366

Phylogenetic affiliation of SSU rRNA genes generated by massively parallel sequencing: new insights into the freshwater protist diversity.  

PubMed

Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2-5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea). PMID:23516585

Taib, Najwa; Mangot, Jean-François; Domaizon, Isabelle; Bronner, Gisèle; Debroas, Didier

2013-03-14

367

Bacterial concrete  

NASA Astrophysics Data System (ADS)

Cracks in concrete are inevitable and are one of the inherent weaknesses of concrete. Water and other salts seep through these cracks, corrosion initiates, and thus reduces the life of concrete. So there was a need to develop an inherent biomaterial, a self-repairing material which can remediate the cracks and fissures in concrete. Bacterial concrete is a material, which can successfully remediate cracks in concrete. This technique is highly desirable because the mineral precipitation induced as a result of microbial activities is pollution free and natural. As the cell wall of bacteria is anionic, metal accumulation (calcite) on the surface of the wall is substantial, thus the entire cell becomes crystalline and they eventually plug the pores and cracks in concrete. This paper discusses the plugging of artificially cracked cement mortar using Bacillus Pasteurii and Sporosarcina bacteria combined with sand as a filling material in artificially made cuts in cement mortar which was cured in urea and CaCl2 medium. The effect on the compressive strength and stiffness of the cement mortar cubes due to the mixing of bacteria is also discussed in this paper. It was found that use of bacteria improves the stiffness and compressive strength of concrete. Scanning electron microscope (SEM) is used to document the role of bacteria in microbiologically induced mineral precipitation. Rod like impressions were found on the face of calcite crystals indicating the presence of bacteria in those places. Energy- dispersive X-ray (EDX) spectra of the microbial precipitation on the surface of the crack indicated the abundance of calcium and the precipitation was inferred to be calcite (CaCO3).

Ramakrishnan, Venkataswamy; Ramesh, K. P.; Bang, S. S.

2001-04-01

368

Bacterial tyrosinases.  

PubMed

Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are important factors in wound healing and primary immune response. Their active site is characterized by a pair of antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a "type 3 copper centre" is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase). The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is still not completely understood, because nothing is known about their tertiary structure. One main reason for this deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-modified monomeric proteins with a low molecular mass of ca. 30kDa. They are secreted to the surrounding medium, where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is essential for the correct expression of the enzyme. This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get more insights in structure, enzymatic reactions and functions of "type 3 copper" proteins in general. PMID:16423650

Claus, Harald; Decker, Heinz

2005-09-06

369

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization  

PubMed Central

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, “Actinobacillus salpingitidis,” and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens.

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-01-01

370

Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.  

PubMed

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens. PMID:14605154

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-11-01

371

Analysis of Internal Transcribed Spacer (ITS) Regions of rRNA Genes in Fungal Communities in a Southeastern U.S. Salt Marsh  

Microsoft Academic Search

The ascomycete community colonizing decaying Spartina alterniflora blades in a southeastern U.S. salt marsh was characterized by analysis of internal transcribed spacer (ITS) regions of fungal rRNA genes. ITS sequences were amplified with ascomycete-specific primers from DNA extracted from S. alterniflora blades at two stages of decay (early and late) and were identified based on sequence analysis of a companion

A. Buchan; S. Y. Newell; J. I. L. Moreta; M. A. Moran

2002-01-01

372

Bacterial start site prediction  

Microsoft Academic Search

With the growing number of completely sequenced bacterial genes, accurate gene prediction in bacterial genomes remains an important problem. Although the existing tools predict genes in bacterial genomes with high overall accuracy, their ability to pinpoint the translation start site remains unsatisfactory. In this paper, we present a novel approach to bacterial start site prediction that takes into account multiple

Sridhar S. Hannenhalli; William S. Hayes; Artemis G. Hatzigeorgiou; James W. Fickett

1999-01-01

373

Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria  

PubMed Central

Background Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV. Methodology/Principal Findings Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel’s clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel’s criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV. Conclusions/Significance The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in Black women. Tools developed in this project can be used to study microbial ecology in diverse settings at high resolution.

Srinivasan, Sujatha; Hoffman, Noah G.; Morgan, Martin T.; Matsen, Frederick A.; Fiedler, Tina L.; Hall, Robert W.; Ross, Frederick J.; McCoy, Connor O.; Bumgarner, Roger; Marrazzo, Jeanne M.; Fredricks, David N.

2012-01-01

374

Bacterial RNA isolation.  

PubMed

In this bacterial RNA isolation protocol, an "RNA-protective" treatment is followed by lysozyme digestion of the peptidoglycan component of the cell wall. EDTA promotes the loss of the outer membrane of Gram-negative bacteria and allows the lysozyme better access to the peptidoglycan. Cells begin to lyse during digestion in hypotonic lysozyme buffer and lysis is completed by sodium dodecyl sulfate (SDS) and hot phenol:chloroform:isoamyl alcohol (PCA) extraction. SDS and hot phenol disrupt membranes, denature protein (including RNase), and strip proteins from RNA. The separation of the organic phase from the aqueous phase is achieved using Phase Lock Gel, an inert material with a density intermediate between the organic and aqueous samples. The sample is split into three phases: from bottom to top, these are phenol and chloroform (organic phase), the inert gel with the interface material, and the aqueous phase with the RNA. The gel acts as a physical barrier between the sample and the organic phase plus interface. Following organic extraction, the RNA is concentrated by ethanol precipitation. PMID:22949721

Ares, Manuel

2012-09-01

375

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

SciTech Connect

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

Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

2008-05-15

376

Bacterial diversity in biofilms formed on condenser tube surfaces in a nuclear power plant.  

PubMed

To elucidate the bacterial diversity in biofilms formed on a condenser tube from a nuclear power plant, 16S rRNA gene sequences were examined using a PCR-cloning-sequencing approach. Twelve operational taxonomic units were retrieved in the clone library, and the estimated species richness was low (13.2). Most of the clones (94.7%) were affiliated with ?-Proteobacteria; Planctomycetes and ?-Proteobacteria were much rarer. Interestingly, except for one clone belonging to Pseudoalteromonas, most of the sequences displayed sequence similarities <97% of those of the closest type strains. Based on 16S rRNA phylogenetic analysis, most bacteria were assigned to novel taxa above the species level. The low species richness and unusual bacterial composition may be attributable to selective pressure from chlorine in the cooling water. To prevent or control bacterial biofilms in cooling circuits, additional studies of the physiology and ecology of these species will be essential. PMID:21058056

Choi, Dong H; Noh, Jae H; Yu, Ok H; Kang, Yeon S

2010-11-01

377

Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system  

PubMed Central

Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ?15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5? external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

Burman, Luke G.; Mauro, Vincent P.

2012-01-01

378

Mutations in 23S rRNA Are Associated with Clarithromycin Resistance inHelicobacter pylori  

Microsoft Academic Search

Twelve clarithromycin-resistantHelicobacter pyloriisolates (100% of resistant isolates examined) from seven different patients each contained an A3G transition mutation within a conserved loop of 23S rRNA. A3G transition mutations at positions cognate with Escherichia coli 23S rRNA positions 2058 and 2059 were identified.Clarithromycin-susceptibleH.pyloriisolatesfrom14differentpatientsdisplayednopolymorphisms in a conserved loop within domain V of 23S rRNA. The study is the first to report mutations

JAMES VERSALOVIC; DEE SHORTRIDGE; KIRSTEN KIBLER; MAMIE V. GRIFFY; JILL BEYER; ROBERT K. FLAMM; S. KEN TANAKA; DAVID Y. GRAHAM; ANDMAE F. GO

379

Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis  

Microsoft Academic Search

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA genes from these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA

Claudio T. Sacchi; Anne M. Whitney; Leonard W. Mayer; Roger Morey; Arnold Steigerwalt; Arijana Boras; Robin S. Weyant; Tanja Popovic

2002-01-01

380

Physical Mapping of the 5S rRNA Multigene Family in Common Wheat  

Microsoft Academic Search

In situ hybridization in conjunction with deletion mapping was used to physically map the 5S rRNA multigene family in Triticum aestivum L. cv. 'Chinese Spring.' Twelve 5S rRNA loci were mapped on chromosomes of homoeologous group 1 (arms 1AS, 1BS, and 1DS) and group 5 (arms 5AS, 5BS, and 5DS). The 5S rRNA loci were mapped (fraction of the distance

Y. Mukai; T. R. Endo; B. S. Gill

381

Epigenetic Programming of the rRNA Promoter by MBD3  

Microsoft Academic Search

Within the human genome there are hundreds of copies of the rRNA gene, but only a fraction of these genes are active. Silencing through epigenetics has been extensively studied; however, it is essential to understand how active rRNA genes are maintained. Here, we propose a role for the methyl-CpG binding domain protein MBD3 in epigenetically maintaining active rRNA promoters. We

Shelley E. Brown; Moshe Szyf

2007-01-01

382

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing  

Microsoft Academic Search

Background Massively-parallel pyrosequencing of hypervariable regions of small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy

Susan M. Huse; Les Dethlefsen; Julie A. Huber; David Mark Welch; David A. Relman; Mitchell L. Sogin

2008-01-01

383

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing  

Microsoft Academic Search

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy

Susan M. Huse; Les Dethlefsen; Julie A. Huber; David Mark Welch; David A. Relman; Mitchell L. Sogin

2008-01-01

384

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge  

Microsoft Academic Search

The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR;\\u000a Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative\\u000a molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits\\u000a of the ATP citrate lyase encoding genes were

James W. Voordeckers; My H. Do; Michael Hügler; Vivian Ko; Stefan M. Sievert; Costantino Vetriani

2008-01-01

385

Multiple Forms of Bacterial Hydrogenases  

PubMed Central

Ackrell, B. A. C. (University of Hawaii, Honolulu), R. N. Asato, and H. F. Mower. Multiple forms of bacterial hydrogenases. J. Bacteriol. 92:828–838. 1966.—Extracts of certain bacterial species have been shown by disc electrophoresis on polyacrylamide gel to contain multiple hydrogenase systems. The hydrogenase enzymes comprising these systems have different electrophoretic mobilities and produce a band pattern that is unique for each bacterial species. Of 20 bacterial species known to possess hydrogenase activity and which were examined by this technique, only the activities of Clostridium tetanomorphum and C. thermosaccharolyticum could be attributed, at pH 8.3, to a single hydrogenase enzyme. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (i.e., RF) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band pattern. The disc electrophoresis technique proved to be 10 times more sensitive than the manometric technique in detecting hydrogenase activity.

Ackrell, B. A. C.; Asato, R. N.; Mower, H. F.

1966-01-01

386

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.  

PubMed Central

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA. Images

Roux, E; Graf, L; Stutz, E

1983-01-01

387

rRNA antitermination functions with heat shock promoters.  

PubMed

Transcription antitermination in the rRNA operons of Escherichia coli requires a unique nucleic acid sequence that serves as a signal for modification of the elongating RNA polymerase, making it resistant to Rho-dependent termination. We examined the antitermination ability of RNA polymerase elongation complexes that had initiated at three different heat shock promoters, dnaK, groE, and clpB, and then transcribed the antitermination sequence to read through a Rho-dependent terminator. Terminator bypass comparable to that seen with sigma(70) promoters was obtained. Lack of or inversion of the sequence abolished terminator readthrough. We conclude that RNA polymerase that uses sigma(32) to initiate transcription can adopt a conformation similar to that of sigma(70)-containing RNA polymerase, enabling it to interact with auxiliary modifying proteins and bypass Rho-dependent terminators. PMID:14563887

Seoh, Hyuk Kyu; Weech, Michelle; Zhang, Ning; Squires, Catherine L

2003-11-01

388

Transformation of tetrahymena thermophila with hypermethylated rRNA genes  

SciTech Connect

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N/sup 6/-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3'', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

Karrer, K.M.; Yao, M.C.

1988-04-01