On the basis of numerical analysis of 100 phenotypic features, the strains of two species, Staphylococcus carnosus and Staphylococcus piscifermentans, were differentiated into two separate phenons corresponding with the macrorestriction patterns of their genomic DNA, as well as with the results of ribotyping and PCR amplification of enterobacterial repetitive intergenic consensus sequences. One of the S. carnosus strains, the F-2 strain, was shown to be marginal, exhibiting the lowest genomic and phenotypic similarity to the S. carnosus type strain DSM 20501T. Two of the strains studied (strains S. carnosus SK 06 and S. piscifermentans SK 05) were phenotypically convergent forming a separate phenon. They were phenotypically similar, even though the genomic DNA of one of them was homologous with that of the S. carnosus type strain, whereas that of the other was homologous with the genomic DNA of the S. piscifermentans type strain. In such cases, fingerprinting methods (particularly macrorestriction analysis and ribotyping) served as important correctives, as they allow phenotypically convergent strains to be distinguished on the basis of their genomic profiles. The results of this paper support the proposal for the new species Staphylococcus condimenti as well as the new subspecies Staphylococcus carnosus subsp. utilis.
The rrs (16S rDNA) gene sequences of nitrogen-fixing endospore-forming bacilli isolated from the rhizosphere of wheat and maize were determined in order to infer their phylogenetic position in the Bacillaceae. These rhizosphere strains form a monophyletic cluster with Paenibacillus azotofixans, Paenibacillus polymyxa and Paenibacillus macerans. Two of them (RSA19 and TOD45) had previously been identified as Bacillus circulans (group 2) by phenotypic characterization (APl 50CH). Evidence for nitrogen fixation by P. azotofixans, P. polymyxa, P. macerans and putative B. circulans strains RSA19 and TOD45 was provided by acetylene-reduction activity, and confirmed by amplifying and sequencing a nifH fragment (370 nt). The phylogenetic tree of nifH-derived amino acid sequences was compared to the phylogenetic tree of rrs sequences. All Paenibacillus nifH sequences formed a coherent cluster distinct from that of related nitrogen-fixing anaerobic clostridia and Gram-positive high-G+C-content frankiae. The nifH gene was neither detected in the B. circulans type strain (ATCC 4513T) nor in the type strains of Bacillus subtilis, Bacillus cereus, Bacillus alcalophilus, Bacillus simplex, Brevibacillus brevis and Paenibacillus validus. Accordingly, nitrogen fixation among aerobic endospore-forming Firmicutes seems to be restricted to a subset of species in the genus Paenibacillus.
The value of rRNA gene RFLP analysis (ribotyping) as a tool for Corynebacterium and Turicella species identification was evaluateed. Seventy-four strains representing 26 different species or subspecies were analysed by BstEll, Smal and Sphl ribotyping. Numerical analysis of the resulting rDNA banding patterns was performed by Dice coefficient correlation in order to establish a database for species identification. In general, most of the strains belonging to the same species clustered together. Interestingly, BstEll clustering of many species followed known phylogenetic lineages. This was not evident with the more heterogeneous Smal and Sphl patterns. The Smal patterns contained a 1800 bp band in the digests of all species studied with the exception of Corynebacterium urealyticum. Sphl digestion resulted in the most heterogeneous patterns. The information provided by all three enzymes was considered essential for the reliable linking of starins of unknown identity with defined species in the database. It is concluded that ribotyping provides an useful tool for screening and charactrization of potentially new Corynebacterium species.
The genotypic and phenotypic diversity among isolates of the Lactobacillus curvatus/Lactobacillus graminis/Lactobacillus sakei group was evaluated by comparing RAPD data and results of biochemical tests, such as hydrolysis of arginine, D-lactate production, melibiose and xylose fermentation, and the presence of haem-dependent catalase. Analyses were applied to five type strains and to a collection of 165 isolates previously assigned to L. sakei or L. curvatus. Phenotypic and RAPD data were compared with each other and with previous DNA-DNA hybridization data. The phenotypic and genotypic separation between L. sakei, L. curvatus and L graminis was clear, and new insights into the detailed structure within L. sakei and L. curvatus were obtained. Individual strains could be typed by RAPD and, after the elimination of similar or identical isolates, two sub-groups in both L. curvatus and L. sakei were defined. The presence or absence of catalase activity further distinguished the two L. curvatus sub-groups. By cloning and sequencing specific RAPD products, pairs of PCR primers were developed that can be used to specifically detect L. curvatus, L. sakei and each of the L. sakei sub-groups.
A phylogenetic investigation was done on the members of the genus Bartonella, based on the DNA sequence analysis of the groEL gene, which encodes the 60 kDa heat-shock protein GroEL. Nucleotide sequence data were determined for a near full-length fragment (1368 bp) of the groEL gene of the established Bartonella species and used to infer intraspecies phylogenetic relationships. Phylogenetic trees were inferred from multiple sequence alignments by using both distance and parsimony methods, which demonstrated an architecture composed of six well-supported lineages. The results are consistent with relationships deduced from recent sequence analysis studies based upon citrate synthase (gltA) and previously observed genotypic and phenotypic characteristics; however, they showed greater statistical support at the intragenus level. This suggests that groEL may be a more robust tool for phylogenetic analysis of Bartonella lineages.
Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.
The DNA relatedness of 17 Leptospira strains isolated from beef cattle in Zimbabwe was determined using the hydroxyapatite method. Similarly to previously speciated African strains, all Zimbabwe isolates belonged to either Leptospira borgpetersenii or Leptospira kirschneri. All serovars within serogroups Pyrogenes (kwale, mombe and a strain closely related to serovar nigeria), Hebdomadis (marondera and mhou), Tarassovi (ngavi) and Sejroe (balcanica and hardjo) were L. borgpetersenii. L. kirschneri contained all stra in serovars of serogroups Icterohaemorrhagiae (zimbabwe), Australis (fugis) Bataviae (paidjan) and Pomona (a strain closely related to mozdok). The species designations of the Zimbabwe fugis and paidjan strains were differe from those of the reference strains of these two serovars, both of which belong to Leptospira interrogans.
The almost complete sequence of the 16S rRNA gene of the Gram-positive polysporogenic bacterium Anaerobacter polyendosporus was determined. This allowed phylogenetic analysis of A. polyendosporus by comparing sequences of the 16S rRNA gene of this bacterium to similar genes of other Gram-positive bacteria. It was shown that this polysporogenic bacterium belongs to the Clostridium cluster I, subcluster A. Phylogenetically, A. polyendosporus is distantly related to another polysporogenic, but non-cultivatable, bacterium, ‘Metabacterium polyspora’ and can be satisfactorily clustered within the saccharolytic clostridia with a low DNA G+C content grouped in subcluster A. A. polyendosporus was most closely related to Clostridium intestinale (94·8% identity of 16S rRNA genes) and Clostridium fallax (93·1 %). Like other members of the Clostridium cluster I, subcluster A, A. polyendosporus possesses such common phenotypic features as a Gram-positive cell wall structure, anaerobiosis, derivation of energy from carbohydrate fermentation yielding butyric acid among other organic acids and the capacity for endogenous spore-formation. However, the scale of evolutionary change in the 16S rRNA gene between A. polyendosporus and phylogenetically related Clostridium species does not correspond to the profound changes in the phenotype of A. polyendosporus. Distinctive phenotypic features of the latter are large cell size, polysporogenesis (up to seven spores per cell), alternative modes of development and an unusual membrane ultrastructure.
Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99·0%, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96·4 to 100%. Sequence similarites between strains representing the two different subspecies ranged from 95·7 to 99·0%. An intervening sequence was identified in certain of the C. hyointestinalis subsp. lawsonii strains. C. hyointestinalis strains occupied two distinct branches in a phylogenetic analysis of the genus Campylobacter, emphasizing the need for multiple strain analysis when using 16S rRNA gene sequence comparisons for taxonomic investigations.
The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning an sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 7 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacill subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accure tool for species-specific identification and phylogenetic analysis of staphylococci.
In order to determine whether morphological criteria are suitable to affiliate myxobacterial strains to species, a phylogenetic analysis of 16S rDNAs was performed on 54 myxobacterial strains that represented morphologically 21 species of the genera Angiococcus, Archangium, Chondromyces, Cystobacter, Melittangium, Myxococcus, Polyangium and Stigmatella, five invalid species and three unclassified isolates. The analysis included 12 previously published sequences. The branching pattern confirmed the deep trifurcation of the order Myxococcales. One lineage is defined by the genera Cystobacter, Angiococcus, Archangium, Melittangium, Myxococcus and Stigmatella. The study confirms the genus status of ‘Corallococcus’, previously ‘Chondrococcus’, within the family Myxococcaceae. The second lineage contains the genus Chondromyces and the species Polyangium (‘Sorangium’) cellulosum, while the third lineage is comprised of Nannocystis and a strain identified as Polyangium vitellinum. With the exception of a small number of strains that did not cluster phylogenetically with members of the genus to which they were assigned by morphological criteria (‘Polyangium thaxteri’ PI t3, Polyangium cellulosum ATCC 25531T, Melittangium lichenicola ATCC 25947T and Angiococcus disciformis An d1), the phenotypic classification should provide a sound basis for the description of neotype species in those cases where original strain material is not available or is listed as reference material.
Fifty-two strains of the yeast species Malassezia pachydermatis were analysed by multilocus enzyme electrophoresis. M. pachydermatis appeared to be genetically heterogeneous. A total of 27 electrophoretic types were identified that could be divided into five distinct groups with different host specificities. The diversity revealed by this electrophoretic method matched remarkably well the reported genetic variability obtained by comparing large subunit rRNA sequences. This study also suggests that genetic exchanges can occur in the anamorphic species M. pachydermatis.