The dnaK operon from Bacillus subtilis and other Gram-positive bacteria with low G+C DNA content contains additional heat-shock genes, including hrcA. The hrcA gene encodes a transcription factor that negatively regulates heatshock genes and is uniformly present in all Gram-positive bacteria studied to date. An hrcA homologue is also present in Synechocystis species, Leptospira interrogans, Chlamydia trachomatis, Caulobacter crescentus and Methanococcus jannaschii, organisms that diverged early on from the common ancestor of all Gram-positive bacteria and Proteobacteria, according to 16S rRNA phylogeny. A partial, protein-based phylogenetic tree, derived using amino acid sequence homology of hrcA proteins from Gram-positive bacteria, is presented here, and the results are compared with the phylogenetic trees generated from 16S rRNA, dnaK and dnaJ sequences. The location of the hrcA gene and the genome organization of the dnaK operon support the division of all Gram-positive bacteria into three major groups: one group contains high-G+C Gram-positive bacteria, and two others contain low-G+C Gram-positive bacteria. Among the Gram-positive bacteria with low G+C DNA content, the results indicate that there is a close phylogenetic relationship between Bacillus species and Clostridium species on the one hand and between Lactococcus lactis and Streptococcus mutans on the other. Streptomyces and Mycobacterium species also exhibited a close relationship. A hierarchical arrangement of Gram-positive bacteria based on HrcA sequences is proposed as an additional refinement of the phylogenetic relationships within this important bacterial group.
The 16S rDNA sequences of the recently described Aeromonas encheleia and Aeromonas popoffii, were determined and compared with data from all known Aeromonas sp. Diagnostic 16S rDNA regions were also sequenced for some strains previously considered as an extension of A. encheleia and a strain of Aeromonas Group 501 (formerly Enteric Group 501). Results indicated that A. encheleia and A. popoffii are phylogenetically separated species as originally described. A. conclusion about HG11 taxonomic status is not recommended until previous discrepancies are clarified by further DNA-DNA hybridization and sequencing studies.
The 16S rRNA sequences of the Korean Borrelia strains 5MT and 9MT, isolated from lxodes nipponensis, showed identities of 99.0-99.1% to that of B. afzelii. The strains were tentatively classified as belonging to the B. afzelii-related group. In this study, Korean isolates, including these strains, were characterized further and compared with recently described new species. These strains generated a RFLP pattern that has not been found previously in RFLP analysis of the 5S-23S rRNA intergenic spacer and the flagellin gene. When phylogenetic trees were constructed, based on the 5S-23S rRNA intergenic spacer, flagellin gene and 16S rRNA sequences, these Korean isolates formed a cluster with the Borrelia strain Am501 isolated from Ixodes columnae in Japan and Borrelia valaisiana strains VS116T and UK isolated from Ixodes ricinus in Europe and were distinguishable from the other species. However, these three groups of strains were divergent from each other in the molecular masses of the putative outer surface protein A (OspA) and in the sequences of the ospA gene. These findings suggest that these Korean isolates and one Japanese isolate are members of B. valaisiana and that OspA of this species is divergent, as is that of Borrelia garinii. This led to the speculation that B. valaisiana strains are adapted to the vector ticks found in each locality.
DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolactic enzymes were distinct at the phylogenetic level, except for malic enzymes of yeast and Escherichia coli which were nearer the malolactic enzymes than the other malic enzymes. The analysis of conserved sites showed several interesting amino acids specific to either malic enzyme or malolactic enzyme.
Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 16S rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDAB and nifH genes, and symbiotic properties. Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15). Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens, respectively, based on their high DNA homologies with the reference strains for these species, their 16S rRNA gene analysis and their phenotypic features. Results of 16S rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium. Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slow-growing, acid-producing characters and the high similarity of their 16S rDNA PCR-RFLP patterns to those of Mesorhizobium species. These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus. The isolates in cluster 12 (Agrobacterium tumefaciens) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes. The possibility of opportunistic nodulation of these isolates is discussed. Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species. The isolates from different host plants in each of clusters 9 and 14 produced different nodDAB RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate). Different patterns were observed among different clusters from both the nod and nif gene hybridization studies. Cross-nodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested.
Recently helicobacter-like organisms have been reported in the pyloric part of the abomasum of calves and adult cattle. Cultivation of these spiral bacteria has not been successful to date. In the present study, comparative 16S rDNA sequence analysis was used to determine the taxonomic position of these bacteria. Seven abomasal biopsies of adult cattle were sampled from different Belgian and Dutch farms. In all samples the presence of helicobacter-like organisms was demonstrated by biochemical, immunohistochemical and electron microscopical data. Bacterial 16S rDNA was amplified by PCR and sequences were determined either by direct or indirect sequence analysis. Pairwise comparisons revealed all sequences to be more than 99% homologous. Phylogenetic analysis placed the organism, corresponding to the reference sequence R2XA, within the genus Helicobacter. A diagnostic PCR assay was designed, differentiating all of the bovine 16S rDNA sequences from Helicobacter and Wolinella species. The low similarity level towards Helicobacter bilis (92·8%), its closest validly named neighbour, indicates that this novel taxon is indeed a novel Helicobacter species. An in situ hybridizatu procedure associated the bovine sequences to the helicobacter-like organism in the abomasum. The name ‘Candidatus Helicobacter bovis’ is proposed for this new abomasal helicobacter from cattle.
The clinical and environmental importance of Stenotrophomonas bacteria requires thorough, molecular studies on their epidemiology and taxonomy. In order to obtain a complete genomic profile of this genus, over 100 Stenotrophomonas maltophilia strains from various origins were investigated by AFLP fingerprinting. A subset of these strains was analysed by DNA hybridization and 16S rDNA sequencing. In contrast to their high phenotypic homogeneity, the strains were found to be very heterogeneous genotypically by AFLP fingerprinting. Nevertheless, ten cores of highly similar strains representing ten genomic groups were observed. The same groups could be retrieved by DNA hybridizations and also, partly, by 16S rDNA sequence analysis. The intergroup DNA similarities were too high to create confident species delineations, neither could the genomic groups be characterized by phenotypic features.
In this study, the phylogenetic relationships between the two biovars and 14 serovars of Ureaplasma urealyticum were studied using the sequences of four different genes or genetic regions, namely: 16S rRNA genes; 16S–23S rRNA gene spacer regions; urease gene subunits ureA, ureB, partial ureC and adjoining regions upstream of ureA, ureA-ureB spacer and ureB-ureC spacer the 5′-ends of the multiple-banded antigen (MBA) genes. U. urealyticum genotypes, based on all four genomic sequences, could be clearly separated into two clusters corresponding with currently recognized biovars 1 and 2. Sequences were generally conserved within each biovar. However, there was heterogeneity within the 5′-end regions of the MBA genes of the four serovas of biovar 1; the sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14 but there were significant differences between the sequences of serovars 3 a 14 and those of serovars 1 and 6. Based on the phylogenetic analysis, support i: given to previous recommendations that the two biovars of U. urealyticum be classified as distinct species, namely U. parvum and U. urealyticum for biovars and 2, respectively. In the future, the relationship between the new species and clinical manifestations of ureaplasma infections should be studied.
An improved pulsed-field electrophoresis program was developed to study differently sized chromosomes within the genus Saccharomyces. The number of chromosomes in the type strains was shown to be nine in Saccharomyces castellii and Saccharomyces dairenensis, 12 in Saccharomyces servazzii and Saccharomyces unisporus, 16 in Saccharomyces exiguus and seven in Saccharomyces kluyveri. The sizes of individual chromosomes were resolved and the approximate genome sizes were determined by the addition of individual chromosomes of the karyotypes. Apparently, the genome of S. exiguus, which is the only Saccharomyces sensu lato yeast to contain small chromosomes, is larger than that of Saccharomyces cerevisiae. On the other hand, other species exhibited genome sizes that were 10–25% smaller than that of S. cerevisiae. Well-defined karyotypes represent the basis for future genome mapping and sequencing projects, as well as studies of the origin of the modern genomes.
The Saccharomyces sp. CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes. The data show that Saccharomyces sp. CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species. Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp. IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule. These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.