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[Variation in the nucleotide sequences of a 16S rRNA gene fragment from cyanobacteria from picoplankton from Lake Baika from natural samples and laboratory cultures].

Nucleotide sequences were determined for the 16S rRNA gene 5T-terminal regions of 29 strains of picoplankton cyanobacteria from the lake Baikal in Russia and four strains from the lake Constance (Bodensee) in Germany. Sequences of a 387-bp region of the gene of cultivated strains designated A, B, C, D, and E had only a few (1-3) substitutions as compared with the 3-27 sequence of the clones obtained earlier by cloning from a pooled sample of Baikalian winter picoplankton. The specific 4-33 sequence obtained earlier from a dominant species of Baikalian picoplankton was not found in cultivated cyanobacteria.

Base Sequence↗
PubMed2003

Phylogenetic relationships among Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

Africa South of the Sahara↗
PubMed2002

Molecular biological methods for studying the gut microbiota: the EU human gut flora project.

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described.

Adult↗
PubMed1998

Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes.

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 experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.

Journal Article↗
PubMed2004

Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers.

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.

Animals↗
PubMed2005

Grouping of streptomycetes using 16S-ITS RFLP fingerprinting.

A total of 463 Streptomyces and Kitasatospora type strains were screened using 16S-ITS RFLP fingerprinting (combined restriction digest using enzymes BstUI and HaeIII). In total, 59 clusters could be delineated, each comprising multiple strains with nearly identical patterns. Good correlation was found in general with phylogeny, as revealed by 16S rDNA sequencing. Most strains assigned to a particular 16S-ITS RFLP cluster were classified into the corresponding 16S sequencing cluster whether a 16S similarity cut-off value of 97 or 98% was used. We conclude that the taxonomic resolution of 16S-ITS RFLP fingerprinting is higher than that of 16S rDNA sequencing; this may provide a tool for reducing the number of laborious DNA-DNA hybridizations necessary for discovering potentially new species within Streptomyces.

Cluster Analysis↗
PubMed1998

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences.

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these gamma-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Bacterial Proteins↗
PubMed1985

Secondary structure of a 345-base RNA fragment covering the S8/S15 protein binding domain of Escherichia coli 16S ribosomal RNA.

A technique for isolating defined fragments of a large RNA has been developed and applied to a ribosomal RNA. A section of the Escherichia coli rrnB cistron corresponding to the S8/S15 protein binding domain of 16S ribosomal RNA was cloned into a single-stranded DNA phage; after hybridization of the phage DNA with 16S RNA and digestion with T1 ribonuclease, the protected RNA was separated from the DNA under denaturing conditions to yield a 345-base RNA fragment with unique ends (bases 525-869 in the 16S sequence). The secondary structure of this fragment was determined by mapping the cleavage sites of enzymes specific for single-stranded or double-helical RNA. The fragment structure is almost identical with that proposed for the corresponding region of intact 16S RNA on the basis of phylogenetic comparisons [Woese, C. R., Gutell, R., Gupta, R., & Noller, H. (1983) Microbiol. Rev. 47, 621-669]. We conclude that this section of RNA constitutes an independently folding domain that may be studied in isolation from the rest of the 16S RNA. The structure mapping experiments have indicated several interesting features in the RNA structure. (i) The largest bulge loop in the molecule (20 bases) contains specific tertiary structure. (ii) A region of long-range secondary structure, pairing bases about 200 residues apart in the sequence, can hydrogen bond in two different mutually exclusive schemes. Both appear to exist simultaneously in the RNA fragment under our conditions. (iii) The long-range secondary structure and one adjacent helix melt between 37 and 60 degrees C in the absence of Mg2+, while the rest of the structure is quite stable.

Bacterial Proteins↗
PubMed1995

Phylogenetic analysis by 16S ribosomal DNA sequence comparison reveals two unrelated groups of species within the genus Ruminococcus.

Phylogenetic analysis of the genus Ruminococcus based on 16S rDNA sequence data showed the genus to be phylogenetically heterogenous. Ruminococcus species fall within the radiation of the Bacillus/Clostridium subphylum of the Gram-positive line of descent. Two distinct and unrelated clusters are recovered. One group contains R. flavefaciens, R. albus, R. bromii, and R. callidus. The second group constitutes R. gnavus, R. hansenii, R. lactaris, R. obeum, R. productus and R. torques which together with some Clostridium species may constitute a new genus.

Bacteria, Anaerobic↗
PubMed2005

Clinical use of 16S rRNA gene sequencing to identify Mycoplasma felis and M. gateae associated with feline ulcerative keratitis.

Routine bacterial cultures of corneal scrapings from seven cats with either ulcerative feline keratitis, keratomalacia, or both yielded colonies which were identified by 16S rRNA gene sequencing as Mycoplasma felis (six cases) and Mycoplasma gateae (one case). Identification of the pathogens allowed the use of less empirical and more organism-specific therapy.

Animals↗
PubMed1999

Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov.

Eubacterium lentum has unique phenotypic characters within the genus Eubacterium. The 16S rRNA sequence of Eubacterium lentum was determined and its phylogenetic position was defined. This micro-organism is a member of the genus Eubacterium but it is not closely related to Eubacterium limosum, the type species of the genus Eubacterium, and is nearer to Collinsella aerofaciens and Coriobacterium glomerans. A PCR-based identification system using species-specific primers designed on the basis of DNA sequences encoding the 16S rRNA of strains of Eubacterium lentum, Collinsella aerofaciens and Coriobacterium glomerans is described. A species-specific primer set can distinguish Eubacterium lentum from Eubacterium limosum or closely related species including Collinsella aerofaciens, Coriobacterium glomerans and Atopobium species. This species-specific PCR method can be used to identify Eubacterium lentum-like species isolated from human faeces. On the basis of the 16S rRNA sequence divergence from Collinsella aerofaciens and Coriobacterium glomerans and the presence of unique phenotypic characters, a new genus, Eggerthella gen. nov., is proposed for Eubacterium lentum, with one species, Eggerthella lenta comb. nov. The type strain of Eggerthella lenta is JCM 9979T.

Bacterial Typing Techniques↗
PubMed1996

Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Bacterial Proteins↗
PubMed2001

Riboprinting and 16S rRNA gene sequencing for identification of brewery Pediococcus isolates.

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.

Base Sequence↗
PubMed2000

[Studies on application for classification and identification in Mycobacterium by analysis of PCR amplification of 16S-23S ribosomal DNA spacer sequences].

The 16S-23S ribosomal DNA spacer sequence of mycobacterium were amplified by PCR. The products were visualized by PAGE, and evaluate the possibility for classification and identification of mycobacterium at gene level. The sensitivity of PCR in annealing temperature 45 degrees C was 500 fg/microL, whereas 50 degrees C was 5 pg/microL. The results showed that: the amplified bands ranging from 300 to 600 bp, most of rapid-growing Mycobacterium and Nonmycobacterium tested have more bands and the bands molecular weights were larger than slow-growing Mycobacterium. The relativity of mycobacterium < 70%, most of them < 50%. This experimental method might be rapid and effective for differentiation of Mycobacterium at species level.

Cluster Analysis↗
PubMed1977

Visualization of ribosomal ribonucleic acid synthesis in a ribonuclease III-Deficient strain of Escherichia coli.

Transmission electron microscopy was used to examine active ribosomal ribonucleic acid (rRNA) genes in two strains of Escherichia coli: N2077, deficient in the enzyme responsible for proper cleavage of the 16S sequence from the elongating nascent rRNA transcript; and N2076, functional in ribonuclease (RNase) III activity, yet otherwise isogenic to N2077. In the strain with wild-type RNase III, double gradients corresponding to a pattern of 16S-cleavage-23S transcription were observed. However, the RNase III-deficient strain exhibited a single ribosomal gradient of approximately the same length as the combined 16S-23S gradients of the wild-type strain. When the rRNA genes were somewhat loosely packed with RNA polymerases, a few of the nascent chains in the ribosomal matrixes of the RNase III-deficient strain were cleaved, but most appeared to be unprocessed. The completed, uncleaved transcripts originating from these gradients are believed to be 30S rRNA molecules recently characterized by biochemical probes.

DNA-Directed RNA Polymerases↗
PubMed2006

Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

Alkalies↗
PubMed2006

Characterization of bacterial populations recovered from the teat canals of lactating dairy and beef cattle by 16S rRNA gene sequence analysis.

Bovine mastitis is of major concern to the dairy industry worldwide. The bovine teat canal is the primary route through which pathogens enter the mammary gland. The microflora of the teat canals of dairy and beef cattle was investigated by analysis of 16S rRNA gene sequences. The goal was to examine the global difference between dairy cattle, which are sanitized on a regular basis, and beef cattle, which receive little sanitary management. A diverse population of 16S rRNA gene sequences was recovered from both the dairy and the beef herd samples, with diversity higher in the beef sample. Analysis revealed the presence of 90 operational taxonomic units (OTUs) among 156 sequences, with 45 OTUs in the dairy sample and 53 OTUs in the beef sample. Only eight OTUs were common to both samples. Members of the classes Clostridia and Bacilli dominated both samples, followed by Actinobacteria and Proteobacteria. The dairy sample contained a cluster (20/80 clones) of Staphylococcaceae members, seven of which were identifiable as coagulase-negative Staphylococcus species. The beef sample was dominated by members of the genus Clostridia, many of which have not been previously cultured. The results suggest that the microorganisms present in the bovine teat canal are more diverse than previously described.

Animals↗