Overestimation of the abundance of sulfate-reducing bacteria in human feces by quantitative PCR targeting the Desulfovibrio 16S rRNA gene

The dominant genus of sulfate-reducing bacteria (SRB) in humans is Desulfovibrio, and quantitative PCR (QPCR) targeting the 16S rRNA gene is often used in assays. We show that the 16S rRNA gene assay overestimated SRB abundance in feces from 24 adults compared to QPCR assays using primers targeting two genes involved in SRB energy metabolism.     

Diversity and origin of Desulfovibrio species: phylogenetic definition of a family

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and "Desulfovibrio multispirans" are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the delta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.     

Estimation of bacterial species phylogeny through oligonucleotide frequency distances

Classification of bacteria is mainly based on sequence comparisons of certain homologous genes such as 16S rRNA. Recently there are challenges to classify bacteria using oligonucleotide frequency pattern of nonhomologous sequences. However, the evolutionary significance of oligonucleotides longer than tetra-nucleotide is not studied well. We performed phylogenetic analysis by using the Euclidean distances calculated from the di to deca-nucleotide frequencies in bacterial genomes, and compared these oligonucleotide frequency-based tree topologies with those for 16S rRNA gene and concatenated seven genes. When oligonucleotide frequency-based trees were constructed for bacterial species with similar GC content, their topologies at genus and family level were congruent with those based on homologous genes. Our results suggest that oligonucleotide frequency is useful not only for classification of bacteria, but also for estimation of their phylogenetic relationships for closely related species.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Sulfate-reducing Bacteria and Mercury Methylation in the Water Column of the Lake 658 of the Experimental Lake Area

Sulfate-reducing bacteria (SRB) appear to be the main mediators of mercury methylation in sediments, which are deemed to be major sites of methylmercury (MMHg) production. However, recent studies have also found significant MMHg formation in the water column of lakes across North America. To investigate the potential involvement of SRB in mercury methylation in the water column of a stratified oligotrophic lake, two of the main families of SRB (Desulfobacteraceae and Desulfovibrionaceae) were quantified by Real-Time Polymerase Chain Reaction of the 16S rRNA gene. MMHg production was measured applying a stable isotope technique using ¹⁹⁸HgCl. Methylation assays were conducted at different water depths and under stimulation with lactate, acetate or propionate and inhibition with molybdate. Desulfobacteraceae and Desulfovibrionaceae16S rRNA gene copies in control samples accounted for 0.05% to 33% and <0.01% to 1.12% of the total bacterial 16S rRNA, respectively. MMHg formation was as high as 0.3 ng L^?1 day^?1 and largest in lactate amended samples. Strain isolation was only achieved in lactate amended media with all isolated strains being SRB belonging to the Desulfovibrio genus according to their 16S rRNA gene sequence. Isolated strains methylated between 0.06 and 0.2% of ¹⁹⁸HgCl per day. Acetate and propionate did not stimulate mercury methylation as much as lactate. Two strains were identified as Desulfovibrio sp. 12ML1 (FJ865472) and Desulfovibrio sp. 12ML3 (FJ865473), based on partial sequences of their 16S rRNA and DSR gene. Methylation assays and bacteria characterization suggest that Desulfovibrionaceae is an important mercury methylators in Lake 658. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.     

Desulfovibrio longus sp. nov., a sulfate-reducing bacterium isolated from an oil-producing well.

A novel type of sulfate-reducing bacteria with unusual morphology was isolated from an oil-producing well in the Paris Basin. The cells of this bacterium, strain SEBR 2582T (T = type strain), are long, thin, flexible rods, contain desulfoviridin, and are physiologically similar to members of the genus Desulfovibrio. On the basis of 16S rRNA sequence data, this strain should be included in the genus Desulfovibrio. However, strain SEBR 2582T differs from other members of this genus morphologically, physiologically, and phylogenetically. Thus, a new species, Desulfovibrio longus sp. nov., is proposed for this organism.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.     

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.     

新疆达坂盐湖沉积土壤嗜盐细菌的定向富集与多样性分析

采集新疆达坂盐湖的沉积土壤样品,以选择性富集培养获得的嗜盐细菌基因组DNA为模板,扩增16SrRNA基因,在此基础上构建嗜盐细菌的16SrRNA基因文库,随机挑选文库中的100个阳性克隆子进行群落结构多样性分析。16SrRNA基因序列分析结果表明:100个克隆分属于细菌域9个属的27个种,其中芽孢杆菌属(Bacillus)为优势菌群(48%),喜盐芽孢杆菌属(Halobacillus)(14%)、盐单胞菌属(Halomonas)(13%)为次优势菌群。分析的阳性克隆子中,10个克隆子与GenBank中已报道16SrRNA基因序列的相似性在88.80%到96.90%之间,可能代表新属或新种。研究结果表明,新疆达坂盐湖沉积土壤的富集培养物中存在种类较为丰富的嗜盐细菌。     

Desulfovibrio brasiliensis sp. nov., a moderate halophilic sulfate-reducing bacterium from Lagoa Vermelha (Brazil) mediating dolomite formation

A novel halotolerant sulfate-reducing bacterium, Desulfovibrio brasiliensis strain LVform1, was isolated from sediments of a dolomite-forming hypersaline coastal lagoon, Lagoa Vermelha, in the state of Rio de Janeiro, Brazil. The cells are vibrio-shaped and 0.30 to 0.45 m by 1.0 to 3.5 m in size. These bacteria mediate the precipitation of dolomite [CaMg(CO₃)₂] in culture experiments. The strain was identified as a member of the genus Desulfovibrio in the -subclass of the Proteobacteria on the basis of its 16S rRNA gene sequence, its physiological and morphological properties. Strain LVform1 is obligate sodium-dependent and grows at NaCl concentrations of up to 15%. The 16S rRNA sequence revealed that this strain is closely related to Desulfovibrio halophilus (96.2% similarity) and to Desulfovibrio oxyclinae (96.8% similarity), which were both isolated from Solar Lake, a hypersaline coastal lake in the Sinai, Egypt. Strain LVform1 is barotolerant, growing under pressures of up to 370 bar (37 MPa). We propose strain LVform1 to be the type strain of a novel species of the genus Desulfovibrio, Desulfovibrio brasiliensis (type strain LVform1 = DSMZ No. 15816 and JCM No. 12178). The GenBank/EMBL accession number for the 16S rDNA sequence of strain LVform1 is AJ544687.     

A highly selective direct method of detecting sulphate-reducing bacteria in crude oil

AIMS: The aim of this work was to develop a highly selective method of detecting sulphate-reducing bacteria (SRB) in crude oil. METHODS: A pair of PCR primers was designed based on an alignment of the nucleotide sequence of the 16S rRNA genes from the Desulfovibrionaceae family. DNA extraction from crude oil was performed by the method using zirconia beads and a stool kit. RESULTS: The PCR specifically detected Desulfovibrio and Desulfomicrobium in a sediment sample. When nucleic acids extracted directly from crude oil were used for the PCR, 16S rRNA genes of Desulfovibrio and Thermodesulforhabdus norvegicus were detected. IMPACT OF STUDY: A simple direct method for detection of the SRB in crude oil using PCR was established.     

A glimpse under the rim – the composition of microbial biofilm communities in domestic toilets

Aim: To determine the microbial composition of biofilms in domestic toilets by molecular means. Methods and Results: Genomic DNA was extracted from six biofilm samples originating from households around Düsseldorf, Germany. While no archaeal 16S rRNA or fungal ITS genes were detected by PCR, fingerprinting of bacterial 16S rRNA genes revealed a diverse community in all samples. These communities also differed considerably between the six biofilms. Using the Ribosomal Database Project (RDP) classifier tool, 275 cloned 16S rRNA gene sequences were assigned to 11 bacterial phyla and 104 bacterial genera. Only 15 genera (representing 121 sequences affiliated with Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes and Proteobacteria) occurred in at least half of the samples or contributed at least 10% of the sequences in a single biofilm. These sequences were defined as ‘typical’ for toilet biofilms, and they were examined in more detail. On a 97% sequence similarity level, these sequences represented 56 species. Twelve of these were closely related to well‐described bacterial species, and only two of them were categorized as belonging to risk group 2. No 16S rRNA genes of typical faecal bacteria were detected in any sample. Virtually all ‘typical’ clones were found to be closely related to bacteria or to sequences obtained from environmental sources, implicating that the flushing water is the main source of recruitment. Conclusion: In view of the great diversity of mostly yet‐uncultured bacteria and the considerable differences between individual toilets, very general strategies appear to be most suited for the removal and prevention of toilet biofilms. Significance and Impact of the Study: For the first time, a molecular fingerprinting and cloning approach was used to monitor the species composition in biofilm samples taken from domestic toilets. Knowledge about the microbial composition of biofilms in domestic toilets is a prerequisite for developing and evaluating strategies for their removal and prevention.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Diversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River (China)

Here we report on the biodiversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in sediment samples from the Xinyi River, Jinagsu Province (China). The biodiversity of aerobic ammonium-oxidizing bacteria in the sediment was assessed using the amoA gene as functional marker. The retrieved amoA clones were affiliated to environmental sequences from freshwater habitats. The closest cultivated relative was Nitrosomonas urea. Anaerobic ammonium-oxidizing (anammox) bacteria were studied using anammox and planctomycete-specific 16S rRNA gene primers. The sediments contained 16S rRNA genes and bacterial cells closely related to the known anammox bacterium Candidatus'Brocadia anammoxidans'. Anaerobic continuous flow reactors were set up to enrich anammox organisms from the sediments. After an adaptation period of about 25 days the reactors started to consume ammonium and nitrite, indicating that the anammox reaction was occurring with a rate of 41-58 nmol cm(-3) h(-1). Community analysis of the enrichments by quantitative fluorescence in situ hybridization showed an increase in the abundance of anammox bacteria from < 1% to 6 +/- 2% of the total population. Analysis of the 16S rRNA genes showed that the enriched anammox organisms were related to the Candidatus'Scalindua' genus.     

Phylogenetic Analyses and Diterpenoid Production by Marine Bacteria of the Genus <Emphasis Type="Italic">Saprospira</Emphasis>

The relationship between 16S rRNA gene sequence-derived phylogeny and the bacterial production of diterpenoids from 18 isolates of marine bacteria belonging to the genus Saprospira was determined. Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified 16S rRNA genes of these isolates indicated four distinct phylotypes. The terpenoid metabolite profiles of each phylotype, determined by liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) analyses, indicated that diterpenoid production was restricted to phylotype A, which included the type specimen S. grandis Gross, and the sole member of the closely related phylotype B. The discovery of two new neoverrucosane diterpenoids produced by phylotype B has also been documented.     

Isolation and characterisation of a novel sulphate-reducing bacterium of the Desulfovibrio genus

A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.     

Natural relationships among sulfate-reducing eubacteria

Phylogenetic relationships among 20 nonsporeforming and two endospore-forming species of sulfate-reducing eubacteria were inferred from comparative 16S rRNA sequencing. All genera of mesophilic sulfate-reducing eubacteria except the new genus Desulfomicrobium and the gliding Desulfonema species were included. The sporeforming species Desulfotomaculum ruminis and Desulfotomaculum orientis were found to be gram-positive organisms sharing 83% 16S rRNA sequence similarity, indicating that this genus is diverse. The gram-negative nonsporeforming species could be divided into seven natural groups: group 1, Desulfovibrio desulfuricans and other species of this genus that do not degrade fatty acids (this group also included "Desulfomonas" pigra); group 2, the fatty acid-degrading "Desulfovibrio" sapovorans; group 3, Desulfobulbus species; group 4, Desulfobacter species; group 5, Desulfobacterium species and "Desulfococcus" niacini; group 6, Desulfococcus multivorans and Desulfosarcina variabilis; and group 7, the fatty acid-oxidizing "Desulfovibrio" baarsii. (The quotation marks are used to indicate the need for taxonomic revision.) Groups 1 to 3 are incomplete oxidizers that form acetate as an end product; groups 4 to 7 are complete oxidizers. The data were consistent with and refined relationships previously inferred by oligonucleotide catalogs of 16S rRNA. Although the determined relationships are generally consistent with the existing classification based on physiology and other characteristics, the need for some taxonomic revision is indicated.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

海水养殖场沉积物中硝酸盐还原菌种群分析

通过对福建省沿海海水养殖场沉积物中参与氮循环的各生理群细菌数量分析 ,发现氨化和硝酸盐还原细菌是优势生理菌群 ,同时 ,表层泥样中的硝酸盐还原菌数量明显高于深层泥样。从该环境中分离获得 106株细菌 ,其中 58株具有硝酸盐还原能力 ,初步鉴定表明它们主要为芽孢杆菌属 (Bacillus)、盐芽孢杆菌属 (Halobacillus)、短芽孢杆菌属 (Brevibacil lus)、动性球菌属 (Planococcus)和动性杆菌属 (Plano