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PubMed2001

Amycolatopsis albidoflavus sp. nov.

The generic position of an actinomycete strain, 'Pseudonocardia sp.' IMSNU 22139, was investigated by the phylogenetic analysis of 16S rDNA sequence. Comparative studies of 16S rDNA sequences indicated that this organism consistently formed a distinct clade within the radiation of the genus Amycolatopsis of the family Pseudonocardiaceae. This organism was also found to have chemotaxonomic properties consistent with those of the genus Amycolatopsis, which were represented by a type IV cell wall (meso-diaminopimelic acid, arabinose and galactose), a major menaquinone of MK-9(H4), a predominant fatty acid of 14-methylpentadecanoic acid, phosphatidylmethylethanolamine as a diagnostic phospholipid (a phospholipid type PII pattern) and DNA base composition of 68.5 mol% G+C. On the basis of physiological properties, cellular fatty acid profiles and its unique phylogenetic position, this organism is readily differentiated from all of the validly described species of the genus Amycolatopsis, and the name Amycolatopsis albidoflavus sp. nov. is proposed for it. The type strain is IMSNU 22139T (= KCTC 9471T = ATCC 53205T).

Actinomycetales↗
PubMed2002

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval.

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.

Archaea↗
PubMed1991

Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.

Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.

Autoradiography↗
PubMed1991

Detection of Mycoplasma bovis and Mycoplasma agalactiae by oligonucleotide probes complementary to 16S rRNA.

The partial sequences of 16S rRNA from Mycoplasma bovis and M. agalactiae were determined by dideoxynucleotide sequencing using reverse transcriptase. Two oligonucleotides complementary to different evolutionary variable regions of 16S rRNA from these two species were synthesized. The oligonucleotides were end-labelled with 32P and used as probes in filter hybridization experiments with different bovine, caprine and ovine mycoplasmas as samples. One of the probes, complementary to a sequence of the V8-region of both M. bovis and M. agalactiae, did not cross-hybridize to any bovine, caprine or ovine mycoplasmas except M. bovigenitalium and M. californicum. This probe is thus not useful for analysis of bovine samples, but can be used for detection of M. agalactiae in samples from goats and sheep, since M. bovigenitalium and M. californicum have never been isolated from these hosts and M. bovis only occasionally. The other probe, complementary to a sequence of the V6-region of M. bovis, gave some cross-hybridization with M. agalactiae but not with bovine mycoplasmas. M. agalactiae has never been isolated from cattle and this probe is therefore useful for rapid screening of bovine samples for M. bovis.

Animals↗
PubMedDate not supplied

Genetic interrelationships of proteolytic Clostridium botulinum types A, B, and F and other members of the Clostridium botulinum complex as revealed by small-subunit rRNA gene sequences.

The phylogenetic interrelationships of members of the Clostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolytic C. botulinum types A, B, and F, and C. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D and C. novyi type A, and iv) type G and C. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the the 'species complex' on the basis of phenotypic criteria.

Base Sequence↗
PubMed2005

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries.

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.

Anti-Bacterial Agents↗
PubMed1998

16S rRNA gene sequences of 'Candidatus Campylobacter hominis', a novel uncultivated species, are found in the gastrointestinal tract of healthy humans.

Although some Campylobacter species are agents of gastroenteritis and periodontal disease in humans, little is known of the variety of campylobacters in the gastrointestinal tract of healthy individuals. This paper provides evidence for the existence of a previously undescribed, uncultivated Campylobacter species that may be a commensal in the healthy human gut. Saliva and faeces from 20 healthy individuals were examined by PCR assays specific for nine species of campylobacter (C. sputorum, C. concisus, C. upsaliensis, C. helveticus, C. lari, C. fetus, C. hyointestinalis, C. jejuni and C. coli) and for the genus as a whole. Genus-specific amplicons were produced from 19 of 20 saliva samples and from 18 of 20 faecal samples. C. concisus species-specific amplicons were produced from 19 of 20 saliva samples and 3 of 20 faecal samples. The faecal samples were all PCR-negative for other Campylobacter species. Three unidentified 16S rRNA Campylobacter genus-specific amplicons of faecal origin were sequenced. Phylogenetic analysis showed that these sequences were 99% similar, and clustered within the genus as a novel group which was termed HS (HS = healthy subject). A PCR primer pair specific for the HS group was designed from the sequence data and used to reexamine the original samples. Although it was not possible to culture the organism from faeces, specific PCR assay detected it in 10 of the 20 faecal samples, but not in any corresponding saliva samples. The authors propose that the source of the amplicons is a previously undescribed and so far uncultivated species, which they term 'Candidatus Campylobacter hominis'.

Adolescent↗
PubMed1994

Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats.

Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerobiosis↗
PubMed2003

Metagenome survey of biofilms in drinking-water networks.

Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.

Bacteria↗
PubMed1991

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.

Animals↗
PubMed2005

Rarity associated with specific ecological niches in the bacterial world: the 'Synergistes' example.

The 'Synergistes' group, which apparently represents an as yet unnamed division of the bacteria, was explored in 93 anaerobic environments (guts, soils, digestors, etc.). From 16S rDNA gene-targeted polymerase chain reaction (PCR) assays, this group appeared to be present in 90% of the anaerobic microbial ecosystems analysed. The phylogeny of 103 16S rDNA sequences from 30 ecosystems showed a strong link between 16S rDNA sequences and given ecosystems. 'Synergistes' 16S rDNA sequences from animal sources (termites, guinea pigs, pigs, birds, etc.) formed clustered phylogenetical groups. 'Synergistes' groups were also associated either with anaerobic digestors and soils or with thermophilic conditions. Sequences available from the DNA database were consistent with the results. These results show the wide diversity of the 'Synergistes' division as well as the specific ecological niche of each 16S rDNA sequences.

Anaerobiosis↗
PubMed2004

The sequence heterogenicities among 16S rRNA genes of Salmonella serovars and the effects on the specificity of the primers designed.

Previously, we have reported a 16S rDNA targeted polymerase chain reaction (PCR) method for the specific detection of Salmonella serovars [J. Appl. Bacteriol. 80 (1996) 659]. The target sites of its primers, i.e. 16SFI and 16SIII, according to the data in GenBank, were found mismatched to the corresponding sequences of some Salmonella serovars, such as those of S. Houten, S. Chingola, S. Bareilly, and S. Weltevreden. Accordingly, a PCR method using a nonspecific primer MINf combined with a primer modified from our 16SFI primer, i.e. the primer MINr, was developed and displayed better detection specificity [Int. J. Food Microbiol. 80 (2003) 67]. In this study, we show the sequence heterogenicity at the primer 16SFI targeting sites for some Salmonella serovars. Thus, the sequence used for designing of PCR primers might be just one of the several possible sequences. Such a situation may lead to the misjudgment on evaluation of the specificity of the primers if this was only based on the data in GenBank. Strains of the above described Salmonella serovars with target sequences from GenBank mismatched to the primer 16SF1 were reidentified and their PCR results were confirmed. Meanwhile, their 16SFI/16SIII primer annealing sites were sequenced and the sequences obtained were found completely and highly homologous to those of 16SFI and complementary to those of 16SIII primer, respectively.

Base Sequence↗
PubMed2003

Multiplex PCR screening of soil isolates for novel Bacillus-related lineages.

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze-heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.

Bacillus↗
PubMed2005

Mitsuaria chitosanitabida gen. nov., sp. nov., an aerobic, chitosanase-producing member of the 'Betaproteobacteria'.

Four strains (3001(T), 2, 12 and 13), which were isolated as chitosanase-producing bacteria from soil from Matsue city (Japan), were studied phenotypically, genotypically and phylogenetically. Based on sequence analysis of 16S rRNA genes, DNA G+C content (67.4-69.2 mol%), quinone type (UQ-8), major fatty acid composition (3-OH 10:0, 3-OH 14:0) and other phylogenetic studies, strains 3001(T), 12 and 13 were found to occupy a separate position in the 'Betaproteobacteria'. Roseateles depolymerans, Rubrivivax gelatinosus and Ideonella dechloratans were their closest neighbours (93-95% 16S rRNA gene sequence similarity). The 16S rRNA gene sequence and other characteristics suggested that strain 2 belonged to the genus Flavobacterium. DNA-DNA hybridization experiments supported the conclusion that strains 3001(T), 12 and 13 were of the same species (72-78% DNA hybridization) and only distantly related to I. dechloratans and R. gelatinosus. It is proposed that strains 3001(T), 12 and 13 represent a novel genus and species for which the name Mitsuaria chitosanitabida gen. nov., sp. nov. is proposed. The type strain of Mitsuaria chitosanitabida is 3001(T) (=IAM 14711(T)=ATCC BAA-476(T)).

Bacteria, Aerobic↗
PubMed2002

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization.

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.

Colony Count, Microbial↗