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PubMed1997

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture.

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.

Base Sequence↗
PubMed1993

Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction.

The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Base Sequence↗
PubMed1996

Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group.

Almost complete (> 96%) 16S rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16S rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalatiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycoplasma bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16S rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16S rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (> 98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.

Animals↗
PubMed1997

Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8.

A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.

Base Sequence↗
PubMed1996

Ehrlichia-like 16S rDNA sequence from wild white-tailed deer (Odocoileus virginianus).

The reservoir hosts of Ehrlichia chaffeensis, etiologic agent of human ehrlichiosis are unknown. Initially, white-tailed deer (WTD) were serologically implicated as possible reservoirs of E. chaffeensis. Subsequent studies showed that WTD were susceptible to infection with E. chaffeensis and that deer-to-deer transmission by a tick vector, Amblyomma americanum, is possible under experimental conditions. To determine if wild WTD were infected with E. chaffeensis, whole blood was collected from 10 deer from Oklahoma and Georgia. All 10 deer had antibodies reactive to E. chaffeensis. Whereas E. chaffeensis was not isolated, restriction enzyme mapping and sequencing of the 16S rDNA gene revealed that a unique Ehrlichia-like agent was present. All 10 deer appeared to be infected with the same agent. We suspect that A. americanum is the vector of this new agent based upon the previously published temporal association between the appearance of E. chaffeensis seropositive WTD and A. americanum. However, the taxonomic and antigenic relationships, geographic distribution, epidemiology, and zoonotic potential of this agent are yet to be determined.

Animals↗
PubMed1993

The phylogeny of the genus Yersinia based on 16S rDNA sequences.

The inter- and intrageneric relationships of the genus Yersinia were investigated by sequence analysis of the 16S rRNA gene. A stretch of approximately 1450 nucleotides was sequenced from representatives of ten of the eleven validly described species. Phylogenetic analysis revealed that yersinae form a coherent cluster within the gamma subgroup of Proteobacteria. The intrageneric relationship was characterized by five sublines with Y. enterocolitica, Y. rohdei, and Y. ruckeri forming separate sublines each represented by a single species. A separate subline was formed by Y. pestis, Y pseudotuberculosis and Y. kristensenii, while Y. mollaretii, Y. intermedia, Y. bercovieri, Y. aldovae, and Y. kristensenii formed a fifth subline. The phylogenetic distinctness of the yersiniae sublines is compared to published phenotypic properties and results of DNA-DNA similarity studies.

Base Sequence↗
PubMed2005

16S rRNA sequences and differences in bacteria isolated from the Muztag Ata glacier at increasing depths.

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2 degrees C or -2 degrees C up to approximately 20 degrees C), although the majority (82%) were psychrotolerant (grew at 2 degrees C or -2 degrees C up to 37 degrees C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were gamma-Proteobacteria, 14.7% were alpha-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.

Aerobiosis↗
PubMed1998

Taxonomic rearrangements of the genera Thiocapsa and Amoebobacter on the basis of 16S rDNA sequence analyses, and description of Thiolamprovum gen. nov.

Complete nucleotide sequences of the 16S rDNAs were determined from Thiocapsa and Amoebobacter species, including all available type strains and some additional isolates. The distance-matrix analysis and the dendrogram for estimating the genetic relationships revealed that the investigated strains were found in two major clusters within the Chromatiaceae. One cluster comprises all Amoebobacter species, Thiocapsa roseopersicina and several isolates related to Thiocapsa roseopersicina. Representatives of the species Amoebobacter roseus, Amoebobacter pendens and Thiocapsa roseopersicina, the so called 'Thiocapsa roseopersicina group', are very closely related, justifying their inclusion into one genus, Thiocapsa, for which an emended description is presented. Amoebobacter purpureus and Amoebobacter pedioformis formed two separate lines of descent with less than 93% (89.6-92.9%) similarity to strains of the 'Thiocapsa roseopersicina group'. Therefore, they will be considered as two separate genera. As a consequence, an emended description is presented for the genus Amoebobacter, with Amoebobacter purpureus as the new type species and A. pedioformis is transferred to Thiolamprovum pedioforme gen. nov., comb. nov. Two species, Thiocapsa pfennigii and Thiocapsa halophila, which have been classified with the genus Thiocapsa because of their morphological properties, were found within another major cluster of the Chromatiaceae and are only distantly phylogenetically related to the first cluster with 88.4-90.6% and 90.4-92.2% sequence similarity, respectively.

Chromatiaceae↗
PubMed1989

[Primary structure of the leading sequence of 16S rRNA gene in Brassica napus chloroplast].

The 840bp leading sequence of 16S rRNA gene (including 140bp 16S rRNA gene) was sequenced. Screening for structure elements common to tRNA reveals a gene coding for tRNA(Val). An open reading frame, and three E. coli RNA polymerase binding sites were found. In front of the open reading frame, the tRNA(Val) gene, and the 16S rRNA gene, a stable stem-loop structure can be formed. We suggested that these stem-loop structures may have some effect on the gene transcription which are located on the inverted repeat sequence in chloroplast.

Base Sequence↗
PubMed1999

Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing.

> Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater.

Journal Article↗
PubMed1996

S Cycling: Characterization of Natural Communities of Sulfate-Reducing Bacteria by 16S rRNA Sequence Comparisons

Past studies of microbial communities responsible for geochemical transformations have been limited by an inability to representatively cultivate, and then identify, the constituent members. Ribosomal RNA sequences, particularly 16S-like rRNAs, provide a measure of phylogenetic relationship that can now be used to examine the structure and diversity of microbial communities. Sulfate-reducing bacteria (SRB) play an important role in the sulfur cycle and the terminal mineralization of organic matter in estuarine and marine environments. Because the Gram-negative mesophilic SRB comprise a phylogenetically coherent assemblage, their communities are well suited to explorations through rRNA sequence-based methodologies. In this study we related molecular biological methods using rRNA probes to geochemical measurements at two different sites. At an unvegetated site in northwest Florida, rates of sulfate reduction were low and SRB rRNA comprised about 5% of the total rRNA extracted from the sediment. The other site, a salt marsh in New Hampshire, had higher rates of sulfate-reduction with SRB rRNA accounting for up to 30% of the total rRNA extracted from the sediment. SRB community structure differed dramatically between the two sites with Desulfobulbus rRNA much less abundant in the unvegetated site than in the salt marsh. The differences in these SRB communities reflect differences in the ecology of their habitats.

Journal Article↗
PubMed2002

[Analysis of 16S rDNA sequences and DNA-DNA hybridization of moderately halophilic bacteria from Xinjiang region].

Based on the previous studies on numerical taxonomy and 16S rDNA PCR-RFLP analysis, the moderately halophilic bacteria isolated from Xinjiang Region constituted a new cluster, and the phylogenetic tree was constructed by comparing with the 16S rDNA sequences of the other moderately halophilic bacteria species. In the phylogenetic tree, most of the reference strains were clustered in a group, and the similarity values of 16S rDNA sequence were above 96%. However, AI-3, Alcanivorax borkumensis and Halobacillus litoralis were clustered in another group, and the similarity value of 16S rDNA sequences between AI-3 and Alcanivorax borkumensis was 96%, and that of 16S rDNA sequences between AI-3 and Halobacillus litoralis was 99%. The results indicated that AI-3 was different from the reference strains in phylogeny. The values of DNA homology in the new cluster were more than 70%, but the value between AI-3 and Halomonas elongata was less than 50%. Thus, the strain AI-3 possibly represent a new moderately halophilic bacteria species.

Base Sequence↗
PubMed2000

16S rDNA sequence analysis of Xylella fastidiosa strains.

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.

DNA, Ribosomal↗
PubMed2004

Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons.

The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but approximately 40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to approximately 2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.

Gene Dosage↗
PubMed2005

Genomic species identification of Acinetobacter of clinical isolates by 16S rDNA sequencing.

INTRODUCTION: This study aims to identify Acinetobacter of clinical isolates from the University of Malaya Medical Centre (UMMC), Kuala Lumpur, to the species level by 16S rDNA sequencing. METHODS: 12 representative Acinetobacter isolates of the UMMC inpatients were randomly picked and used for the study. The 16S rDNA sequences were determined and phylogenetic relationships to all known Acinetobacter species were established. RESULTS: Based on the 16S rDNA sequences, all the UMMC isolates were identified as Acinetobacter baumannii. The isolates shared a common ancestral lineage with the prototypes Acinetobacter baumannii DSM30007 and DSM30008 with 99-100 percent sequence similarities. The isolates could be differentiated into two groups by a single nucleotide difference (thymine-cytosine) within the 16S rRNA sequence. Three different genotypes, 1, 3 and 4, were recognised using REP-PCR. CONCLUSION: The previously uncharacterised Acinetobacter isolates from the UMMC were identified by their 16S rDNA sequences as Acinetobacter baumannii. The isolates were distinguished into at least three different genotypes by REP-PCR genotyping. These findings confirmed for the first time the presence of Acinetobacter baumannii of different genotypes among patients at UMMC.

Acinetobacter baumannii↗
PubMed2004

Phylogenetic analysis of partial bacterial 16S rDNA sequences of tropical grass pasture soil under Acacia tortilis subsp. raddiana in Senegal.

We used direct recovery of bacterial 16S rRNA gene sequences to investigate the bacterial diversity under Acacia tortilis subsp. raddiana, a legume tree naturally growing in the dry land part of Senegal (West Africa). Microbial DNA was purified directly from soil samples and subjected to PCR with primers specific for bacterial 16S rRNA gene sequences. 16S rDNA clone libraries were constructed from two soil samples taken at two dates, i.e. June 25th 1999 (dry season) and August 28th 1999 (rainy season) at depths of 0.25-0.50 m and at 3 m distance from the stem. The 16S rDNA of 117 clones was partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity (100 phylotypes). Comparative sequence analysis of these clones identified members of the Gammaproteobacteria (35% of the phylotypes) as the most important group, followed by the Firmicutes division with 24%. Alphaproteobacteria, Betaproteobacteria, Acidobacteria and Actinobacteria were found to be less represented. Our data suggest that bacterial communities under Acacia tortilis subsp. raddiana might differ according to the season. The relative compositions of the populations is different in both samples: the Acidobacteria are present in a much higher percentage in the dry season than in the rainy season sample while the inverse effect is observed for the members of the other groups. Within the Gammaproteobacteria we found a shift between the dry season and the rainy season from pseudomonads to Acinetobacter and Escherichia related organisms.

Acacia↗
PubMed1982

The complete nucleotide sequence of 16S ribosomal RNA gene from tobacco chloroplasts.

The complete nucleotide sequence of 16S ribosomal RNA gene from tobacco chloroplasts has been determined. This nucleotide sequence has 96% homology with that of maize chloroplast 16S rRNA gene and 74% homology with that of Escherichia coli 16S gene. The 3' terminal region of this gene contains the sequence ACCTCC which is complementary to sequences found at the 5' termini of prokaryotic mRNAs. The large stem and loop structure can be constructed from the sequences surrounding the 5' and 3' ends of the 16S gene. These observation demonstrate the prokaryotic nature of chloroplast 16S rRNA.

Base Sequence↗
PubMed1990

16S rRNA sequences reveal uncultured inhabitants of a well-studied thermal community.

Molecular methods are beginning to reveal inhabitants of natural microbial communities which have never before been cultured. Our approach involves selective cloning of naturally occurring 16S rRNA sequences as cDNA, and comparison of these sequences to a database which includes 16S rRNA sequences of isolated community members. We provide here an overview of the method and its potential for community analysis. A 16S rRNA sequence retrieved from the well-studied hot spring cyanobacterial mat in Octopus Spring (Yellowstone National Park) is shown as an example of one contributed by an uncultured member of the community.

Base Sequence↗