Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.     

Conservation of the response regulator gene gacA in Pseudomonas species

The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Isolation and identification of equol-producing bacterial strains from cultures of pig faeces.

Transformation of daidzein to equol was compared during fermentation of three growth media inoculated with faeces from Erhualian piglets, but equol was produced from only one medium, M1. Two equol-producing strains (D1 and D2) were subsequently isolated using medium M1. Both strains were identified as Eubacterium sp., on the basis of morphological and physiological characteristics, and 16S rRNA gene sequence analysis showed that strains D1 and D2 were most closely related to previously characterized daidzein-metabolizing bacteria isolated from human faecal and rumen samples, respectively. This suggests that the ability to metabolize daidzein can be found among bacteria present within the mammalian intestine. The results provided the first account of conversion of daidzein directly to equol by bacterial species from farm animals. These strains may be of importance to the improvement of animal performance, and the use of medium M1 could provide a simple way to isolate bacterial strains capable of transforming daidzein into equol.     

Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise)

A diffusely venting chimney spire from the East Pacific Rise (9 degrees N) was analysed by petrographic thin sectioning and 16S rRNA gene cloning and sequencing in parallel, to correlate microbial community composition with mineralogy and inferred in situ conditions within the chimney mineral matrix. Both approaches indicated a zonation of the chimney spire into distinct microhabitats for different bacteria and archaea. The thermal gradient inferred from the mineral composition and porosity of the chimney was consistent with the distribution of bacterial and archaeal phylotypes in the chimney matrix. A novel phylogenetic lineage of euryarchaeota was found that co-occurred with clones related to cultured hyperthermophilic archaea. A few phylotypes related to mesophilic bacteria were found in the hot core of the chimney, indicating that seawater influx during retrieval and cooling of these highly porous structures can entrain microorganisms into chimney layers that are not their native habitat.     

High genetic and physiological diversity of sulfate-reducing bacteria isolated from an oligotrophic lake sediment

The community structure of sulfate-reducing bacteria in littoral and profundal sediments of the oligotrophic Lake Stechlin (Germany) was investigated. A collection of 32 strains was isolated from the highest positive dilutions of most-probable-number series, and their partial 16S rRNA gene sequences and genomic fingerprints based on ERIC (enterobacterial repetitive intergenic consensus)-PCR were analyzed. The strains fell into eight distinct phylogenetic lineages, and the majority (70%) showed a close affiliation to the genus Desulfovibrio. Most of the remaining strains (22%) were related to the gram-positive Sporomusa and Desulfotomaculum groups. A high redundancy of 16S rRNA gene sequences was found within several of the phylogenetic lineages. This low phylogenetic diversity was most pronounced for the subset of strains isolated from oxic sediment layers. ERIC-PCR revealed that most of the strains with identical 16S rRNA gene sequences were genetically different. Since strains with identical 16S rRNA gene sequences but different genomic fingerprints also differed considerably with respect to their physiological capabilities, the high diversity detected in the present work is very likely of ecological relevance. Our results indicate that a high diversity of sulfate-reducing bacterial strains can be recovered from the natural environment using the established cultivation media. Received: 20 April 1998 / Accepted: 12 June 1998     

Transmission of nephridial bacteria of the earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Detection and Quantification of Bacteria Involved in Aerobic and Anaerobic Ammonium Oxidation in an Ammonium-Contaminated Aquifer

The aerobic and anaerobic ammonium-oxidizing bacterial guilds were studied from two multilevel samplers in an ammonium-contaminated aquifer in the UK. By end point polymerase chain reaction (PCR), the presence of betaproteobacterial ammonium-oxidizing bacteria and anaerobic ammonium-oxidizing (anammox) planctomycetes was demonstrated. The sequences of cloned anammox-specific PCR fragments had close relationships with known anammox strains. Real-time PCR was subsequently used to determine the relative size of betaproteobacterial ammonium-oxidizing bacteria and anammox bacterial guilds in relation to the whole bacterial community, showing large differences between the two multilevel samplers. The depth profiles of the guild sizes correlated well with the profiles of the major geochemical parameters such as ammonium, nitrate, nitrite, and oxygen. A maximum of, 24% of anammox planctomycetes, 16S rRNA gene copies within the total bacterial, 16S rRNA gene copies in one of the boreholes indicated that the anammox process could have an important contribution in the natural attenuation of the ammonium plume at the site.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico

PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale. Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates. Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups. Two isolates were closely related to M. loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M. amorphae, M. plurifarium and M. huakuii. However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences. Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene. A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene. These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.     

Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.     

Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates

A total of 319 bacterial strains isolated from the surfaces of seaweeds and invertebrates were tested for their effects on settlement of Ulvalactuca spores and Hydroidesezoensis larvae in laboratory bioassays. Of the 192 bacterial strains isolated from the surfaces of seaweeds 63 isolates were shown to be inhibitory against the settlement of algal spores and 62 isolates were inhibitory against larval settlement. Thirty-seven percent of the 127 bacterial strains isolated from the surfaces of marine invertebrates were shown to be inhibitory against algal spores and larval settlement. The strain CI4 isolated from an adult Cionaintestinalis showed the strongest inhibitory effect and was identified as Pseudoalteromonas sp. via 16S rRNA gene sequencing. The high proportions of host associated bacteria producing antifouling compounds suggest that these bacteria may help the host organism in the defense against fouling.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe.

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.     

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.     

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

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

Rapid differentiation of bacterial species by high resolution melting curve analysis

Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it’s application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.