- Volume 48, Issue 3, 1998

The 16S-23S internally transcribed spacer (ITS) sequences were analysed to clarify inter-and intraspecific relationships among strains of the genus Nocardioides and the relationship between two Aeromicrobium species. The 16S-23S ITS regions from 33 Nocardioides strains, two Aeromicrobium species and Terrabacter tumescens were sequenced directly after polymerase chain reaction (PCR) amplification and λ exonuclease treatment. The genomes of some Nocardioides strains included two types of 16S-23S ITS sequences. The sizes of the 16S-23S ITS sequences of Nocardioides strains ranged from 328 to 539 bp. The 16S-23S ITS sequences of Aeromicrobium erythreum NSP37T, Aeromicrobium fastidiosum NSP38Tand T. tumescens NSP39Twere 349, 355 and 386 bp long, respectively. Nucleotide similarity among 16S-23S ITS sequences of Nocardioides albus strains and of Nocardioides simplex strains was 84·1-100% and 97·7-100%, respectively. The 16S-23S ITS sequence of Nocardioides luteus was identical to that of “Nocardioides fulvus” NSP32Tand was only 1 bp different from that of “Nocardioides fulvus” strains. However, the 16S-23S ITS sequences of “N. fulvus” NSP32Tshowed only a low degree of similarity to “N. fulvus“ NSP32T(54·8%). The degree of 16S-23S ITS similarity between N. luteus NSP20Tand N. albus strains ranged from 85 to 93%. The mean nucleotide similarity values between the type strains of validly described Nocardioides species were highly divergent at 68·1±16·8%. The two Aeromicrobium species showed a level of 16S-23S ITS similarity of 71·2%. In this study, 16S-23S ITS sequences of the members of the genera Nocardioides and Aeromicrobium were useful for inferring the relationships between closely related strains and species. However, they were not found to be appropriate for elucidating the phylogenetic relationships between distantly related organisms at the genus level.

Based on the sequence data of 23S rRNA of Staphylococcus carnosus, Staphylococcus piscifermentans, Staphylococcus aureus and Staphylococcus epidermidis, species-specific probes were constructed. Their application revealed a heterogeneity within 18 strains previously identified as S. carnosus. Strains of this group were selected, and their 23S rRNA sequence was determined. It was revealed that the strains of S. carnosus can be placed in at least three sub-groups. This grouping was supported by physiological data and DNA-DNA similarity studies. Based on these results, we propose the new species Staphylococcus condimenti sp. nov. The type strain is S. condimenti F-2T (= DSM 11674T). The phylogenetic position of the new species within the radiation of other staphylococcal strains is reflected by a 16S rRNA-based tree. Furthermore, it is proposed to designate the new subspecies of Staphylococcus carnosus Schleifer and Fischer 1982, Staphylococcus carnosus subsp. utilis subsp. nov. The type strain of S. carnosus subsp. utilis is SK 11T(= DSM 11676T).

The population genetics of Serpulina pilosicoli and its molecular epidemiology in villages in the Eastern Highlands province of Papua New Guinea were investigated. Multilocus enzyme electrophoresis (MLEE) was used to analyse 164 isolates from humans and animals. These were divided into 33 electrophoretic types (ETs), four of which contained 65% of the isolates. The mean genetic diversity (n = number of ETs) for 145 human isolates was 0·18, and the mean number of alleles at five polymorphic loci was 2·6. The species appeared to be recombinant, as there was a lack of linkage disequilibrium, and 25% of all the possible combinations of alleles was present in the population. PFGE analysis using the enzymes Mlul and Sall divided 157 of the isolates into 99 PFGE types, demonstrating the existence of considerable strain diversity in a geographically restricted area. The two techniques were in excellent agreement; however, PFGE was more discriminatory for strain typing than was MLEE. Nine out of 19 (47·4%) culture-positive individuals were colonized by the same PFGE type of S. pilosicoli when retested after 6 weeks. For three individuals, the PFGE profiles of the second isolate differed from the first in only one or two DNA bands, while the other seven individuals were colonized with distinct PFGE types on each occasion. In two cases, strains with the same PFGE pattern were isolated from humans and dogs, suggesting that cross-species transmission of S. pilosicoli may occur naturally and that the infection can be zoonotic.

Strain C1Tis an anaerobic spirochaete that causes intestinal disease in chickens. Multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons have indicated that this spirochaete is a Serpulina strain. In these investigations, various phenotypic and genomic properties useful for establishing a taxonomic identity for strain C1Twere studied. As determined by electron microscopy, cells of the spirochaete measured 8-11 x 0·22-0·34 µm and had a typical spirochaete ultrastructure. Each cell had 22·30 flagella. C1Tcells formed weakly β-haemolytic colonies on trypticase soy agar plates containing 5% bovine blood. The spirochaete reached maximum population densities of 109cells ml-1with a 2·4 h population doubling time in brain heart infusion broth containing 10% calf serum (BHIS broth). C1Tcultures in BHIS broth were positive in tests for hippurate hydrolysis and negative for indole production. Glucosamine, N-acetylglucosamine, glucose, fructose, maltose and mannose were growth substrates for the spirochaete in heart infusion broth containing 7% calf serum (HS broth). During growth in HS broth beneath an O2/N2 (1:99) atmosphere, cells of the spirochaete consumed O2 and glucose and produced H2, CO2, acetate, butyrate and ethanol. Strain C1TDNA had a G+C content of 24·6 mol%. Based on DNA-DNA hybridization analyses, the DNA of strain C1Texhibited 24·39% relative reassociation with DNA of Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Serpulina murdochii and Serpulina intermedia. These results indicate that chicken spirochaete strain C1Thas many phenotypic properties common to Serpulina species and, based on DNA hybridization analysis, represents a unique Serpulina species. For this new species the name Serpulina alvinipulli is proposed, for which the type strain is C1T(= ATCC 51933T).

Nine different strains, CTCBS1Tto CTCBS9, were identified to be the causative agents of black spots on the surface of raw cured meat products. The formation of black spots under aerobic conditions is reproducible upon reinoculation of meat products with any of these strains, indicating that they are the causative agent. The strains were Gram-negative, catalase-positive and obligately aerobic rods. The G+C content of DNA of strain CTCBS1Tis 56·0±0·3 mol%. The content of non-polar main fatty acids were 16:0, 16:1, 18:1 and 19:0 cyc. Its phylogenetic position was elucidated by comparative sequence analysis of the 16S rRNA gene. Overall sequence similarity to other bacteria does not exceed 93·3%. Isolate CTCBS1Tclustered phylogenetically within the β-subclass of the Proteobacteria and is closely related to members of Halomonas (90·5-91·9%) and to Zymobacter palmae (93·3%). A genetic homogeneity of the nine strains was demonstrated by M13 random amplified polymorphic DNA-PCR, whereas differentiation from other genera, e.g. Zymobacter and Pseudomonas, could easily be achieved by their chemotaxonomic characteristics. Taxonomic data revealed the status of a separate genus for which the name Carnimonas gen. nov., sp., nov. is proposed. Despite chemotaxonomic and physiological similarities, the new genus is at present not a member of the family Halomonadaceae because of the lack of two out of 15 descriptive 16S rRNA signature sequences. The first member of the new genus is Carnimonas nigrificans. The use of a specific, 16S rRNA-targeted oligonucleotide primer allowed the identification of all nine strains of C. nigrificans in a PCR assay. Toxicological studies showed no pathogenic potential for C. nigrificans strain CTCBS1T(CECT 4437T).

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate S02T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huautlense is significant because in the reconstruction of the phylogeny of R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum.

A thermophilic, anaerobic, strictly autotrophic, sulphur-reducing bacterium, designated BSAT(T = type strain), was isolated from a deep-sea hydrothermal chimney sample collected at the mid-Atlantic ridge. Gram-negative cells occurred singly or in pairs as small highly motile rods. Spores were not observed. The temperature range for growth was 40 to 75°C, with an optimum at 70 °C. The pH range for growth at 70 °C was from 4·4 to 7·5, with an optimum around 6·0. The sea salt concentration range for growth was 15-70 g I-1with an optimum at 35 g I-1. Elemental sulphur, thiosulphate and sulphite were reduced to hydrogen sulphide. Sulphate and cystine were not reduced. The G+C content of the genomic DNA was 35 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the strain was a member of the domain Bacteria and formed a branch that was almost equidistant from members of the orders Aquificales and Thermotogales. The new organism possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Desulfurobacterium gen. nov., of which Desulfurobacterium thermolithotrophum sp. nov. is the type species. The type strain is BSAT(= DSM 11699T).

Dr Robert Smibert from the Virginia Polytechnic Institute, USA, isolated and collected over 200 strains of oral treponemes over a 20-year period. Dr Smibert, Dr W. E. C. Moore and Dr L. V. Moore separated these isolates and reference strains into different groups on the basis of cellular fatty acid analysis. In this study, the 16S rRNA genes were sequenced for 47 strains that were representative of these groups. Five distinct species were identified on the basis of 16S rRNA sequence comparisons; two of these species are newly named and three have not yet been characterized. The first species, designated Treponema Smibert-1, was represented by the single strain D4B-1 and was later identified as the newly described Treponema maltophilum. However, strain D4B-1 possessed a different flagellar arrangement to that of T. maltophilum. The second species, Treponema Smibert-2, was represented by nine isolates that possessed identical 16S rRNA gene sequences. The closest relatives of this species were Treponema Smibert-3 and Treponema Smibert-4 at approximately 90% sequence similarity. Within Treponema Smibert-2, there was no correlation between phylogenetic analysis and cellular fatty acid analysis since six different cellular fatty acid groups represented the nine strains. Treponema Smibert-3 (strain D36ER-1) and Treponema Smibert-4 (D62CR-12) were each represented by only a single strain and were closely related to each other at 98% sequence similarity. Strain D36ER-1 of Treponema Smibert-3 was identified as belonging to the not-yet-cultivated phylotype 20 [Choi, B. K., Paster, B. J., Dewhirst, F. E. & Göbel, U. B. (1994). Infect Immun 62,1889-1895]. Strain D62CR-12 of Treponema Smibert-4 was nearly identical in sequence to the newly described Treponema amylovorum. The fifth species, Treponema Smibert-5, was represented by a single strain, D120CR-1, and was closely related at about 98% sequence similarity to the three subspecies of Treponema socranskii. The phylogenetic analyses of strains of Treponema vincentii and of subspecies of T. socranskii are also reported. The closest oral relatives of T. vincentii were Treponema medium at 98·7% sequence similarity and Treponema denticola at 91·5% sequence similarity. T. socranskii subspp. socranskii, buccale and paredis formed three separate phylogenetic branches with sequence similarities of about 98% to each other. The closest relative of the subspecies of T. socranskii and of Smibert-5 was Smibert-2 at about 86% sequence similarity. Historic reference strains Fuji, "Treponema ambigua", Fm, lchelson-2, N-39, TD2, TRRD, MRB, IPP, Jethro and T32A, as well as an unknown strain designated only as Treponema oralis, were identified as strains of T. denticola. Reference strains Fuji, Jethro, T32A and IPP plus three isolates of the Smibert collection were also contaminated with a mycoplasma as determined by 16S rRNA comparative analysis. Consequently, spirochaetal cultures should be screened for mycoplasmas. There are presently at least ten species of cultivable oral species of Treponema with the cut-off for separate species

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey. J. V., Birtles, R. J. & Saunders, N. A. (1995). J Clin Microbiol 33, 2377-2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521-1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114-132 of E. coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after HintI restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Analysis of the 16S rDNA sequences of species currently assigned to the genus Herpetosiphon revealed intrageneric phylogenetic heterogeneity. The thermotolerant freshwater species Herpetosiphon geysericola is most closely related to the type species Herpetosiphon aurantiacus in the Chloroflexus subdivision of the green non-sulfur bacteria. The marine species Herpetosiphon cohaerens, Herpetosiphon nigricans and Herpetosiphon persicus, on the other hand, were found to form a cluster with the sheathed bacterium Haliscomenobacter hydrossis in the Saprospira group of the Flexibacter-Bacteroides-Cytophaga (FBC) phylum. A proposal is made to transfer these marine species to the genus Lewinella gen. nov. as Lewinella cohaerens comb. nov., Lewinella nigricans comb. nov. and Lewinella persica comb. nov. The marine sheathed gliding bacterium Flexithrix dorotheae was also found to be a member of the FBC phylum but on a separate phylogenetic line to the marine herpetosiphons now assigned to the genus Lewinella.

The 16S rRNA gene sequences of 19 strains, 11 strains representing validated Aureobacterium or Microbacterium species and eight strains of non-valid species or isolates, were determined. These sequences were aligned with the sequences of other validated Aureobacterium and Microbacterium species and related actinobacteria. A comparative sequence analysis of 43 strains revealed that the species of the genera Aureobacterium and Microbacterium form a monophyletic association in which species of both genera are intermixed. The high similarity in phylogenetic properties found in the species within both genera and the close relationship in physiological and chemotaxonomic features other than the diamino acid in the cell wall, provided strong evidence that the genera Aureobacterium and Microbacterium should be unified. An emended genus Microbacterium is proposed for the two combined genera. The following validated Aureobacterium species were combined to the genus Microbacterium: Aureobacterium arabinogalactanolyticum to Microbacterium arabinogalactanolyticum, Aureobacterium barkeri to Microbacterium barkeri, Aureobacterium esteraromaticum to Microbacterium esteraromaticum, Aureobacterium flavescens to Microbacterium flavescens, Aureobacterium keratanolyticum to Microbacterium keratanolyticum, Aureobacterium liquefaciens to Microbacterium liquefaciens, Aureobacterium luteolum to Microbacterium luteolum, Aureobacterium saperdae to Microbacterium saperdae, Aureobacterium schleiferi to Microbacterium schleiferi, Aureobacterium terrae to Microbacterium terrae, Aureobacterium terregens to Microbacterium terregens, Aureobacterium testaceum to Microbacterium testaceum, and Aureobacterium trichothecenolyticum to Microbacterium trichothecenolyticum.

A collection of 94 Vibrio isolates closely related to Vibrio harveyi, together with named reference and type strains, were investigated for phenotypic and genotypic properties. Using amplified fragment length polymorphism (AFLP), nine clusters were recognized. The largest cluster (n = 36), considered to be the bona fide V. harveyi group, contained the type strains of V. harveyi and Vibrio carchariae and most of the strains isolated from fish. The type strains of all other species, including Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio campbellii and Vibrio natriegens, clustered outside this group. By ribotyping, V. harveyi and V. carchariae patterns were very similar, insofar as they shared most bands. The V. campbellii type strain had several bands in common with the type strains of both V. harveyi and V. carchariae, whereas the other species were clearly distinct from these three species. DNA-DNA hybridization experiments showed 88% DNA binding between the type strains of V. harveyi and V. carchariae, whereas the DNA binding between V. harveyi and V. campbellii was 40%. Although the delineation of the species V. harveyi is still uncertain, the authors propose, on the basis of a number of tests, to delineate a core of V. harveyi strains which contained the type strains of both V. harveyi and V. carchariae. It is concluded that V. carchariae is the junior synonym of V. harveyi.

The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331Ta result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331Twas in a separate cluster. The LMG 3301 and the LMG 3331Tclusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T= NCTC 12171T= CNS 2-75T).

An aerobic, polarly flagellated marine bacterium that produces a prodigiosinlike pigment was isolated from the red-spotted culture beds of Laminaria japonica. Five isolates had unique bacteriolytic activity for both Gram-positive and -negative bacteria, which had never been observed among Alteromonas or related species. The isolates were identified as the causative agent of red spot disease of L. japonica seeds. The phenotypic features of the isolates were similar to these of Pseudoalteromonas rubra ATCC 29570T, but they could be differentiated using 10 traits (growth at 37·C, requirement for organic growth factors, bacteriolytic activity, utilization of sucrose, N-acetylglucosamine, fumarate, succinate, d-galactose, l-proline and acetate). The G+C content of DNAs from the isolates was 44-46 mol%. The isolates constitute a new species, distinct from the other Alteromonas and Pseudoalteromonas species, as shown by DNA-DNA hybridization experiments and phylogenetic clustering of 16S rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica sp. nov. (type strain = IAM 14595T) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided.

A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge. The cells were Gram-negative, catalase-and oxidase-positive curved rods and very motile. They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors. The G+C content was 65 mol%. Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems. However, a phylogenetic analysis based on the 16S rRNA sequence showed that the aerobic denitrif could not be assigned to any currently recognized genus. For these reasons a new genus and species, Microvirgula aerodenitrificans gen. nov., sp. nov., is proposed, for which SGLY2Tis the type strain.

A novel, obligately anaerobic bacterium capable of hydrolysing lipids was isolated from a tropical anaerobic lagoon receiving waste water from an edible oil mill. The isolate had many characteristics similar to those of members of the genus Selenomonas. The isolate showed lipolytic activity on tributyrin, triolein and groundnut oil in qualitative plate clearance assays, which has not been reported for the type strain of the genus Selenomonas. It did not require n-valerate supplementation for growth on glucose. Acetate and propionate were the only volatile fatty acids produced from glucose fermentation with propionate as the major end product. The isolate could grow optimally at pH 6.8 and at a temperature of 40 °C. It could tolerate NaCI concentrations of up to 40 g l-1. The G+C content of the DNA was 40 mol% as determined by thermal denaturation analysis. Comparison of partial 16S rRNA gene sequences revealed that the isolate was most closely related to genus Selenomonas with 91% sequence similarity (250 bp compared) to Selenomonas ruminantium strain GA 192. On the basis of the results obtained in the present investigation, it is suggested that a new species of Selenomonas should be created for this novel isolate and the name Selenomonas lipolytica is proposed for this new species. The type strain is strain CF1BT(= MCMB 505T).

The 16S rDNA sequence of Rhodospirillum mediosalinum was determined and compared with corresponding sequences from other spiral-shaped purple non-sulfur bacteria classified as or related to the genus Rhodospirillum in the α subclass of the Proteobacteria. Sequence similarities separate the currently recognized Rhodospirillum species into five different groups with no more than 91% sequence similarity, clearly indicating the necessity to recognize these groups as different genera. Major diagnostic properties of these bacteria are compared and new genera Phaeospirillum gen. nov., Roseospira gen. nov., Rhodothalassium gen. nov. and Rhodovibrio gen. nov. are described with the species Phaeospirillum fulvum comb, nov., Phaeospirillum molischianum comb. nov., Rhodovibrio salinarum comb. nov., Rhodovibrio sodomensis comb. nov., Rhodothalassium salexigens comb. nov. and Roseospira mediosalina comb. nov. The genus Rhodospirillum is represented by Rhodospirillum rubrum and Rhodospirillum photometricum and an emended description of this genus is also given.

A new subspecies, Staphylococcus hominis subsp. novobiosepticus, isolated from human blood cultures, a wound, a breast abscess and a catheter tip, is described on the basis of a study of 26 strains isolated between 1989 and 1996. DNA-DNA reassociation reactions, conducted under stringent conditions, and macrorestriction pattern analysis demonstrated that these strains are closely related to previously characterized S. hominis strains isolated from human skin and clinical specimens, but are significantly divergent. S. hominis subsp. novobiosepticus can be distinguished from S. hominis (now named S. hominis subsp. hominis) by its combined characteristics of novobiocin resistance and failure to produce acid aerobically from D-trehalose and N-acetyl-D-glucosamine. Furthermore, all 26 strains of the new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin, and were either resistant or had intermediate resistance to methicillin and gentamicin. Most strains were also resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprofloxacin. Based on a comparison of the sequences of a 1001 bp mecA amplification product from reference methicillin-resistant staphylococci, the mecA gene present in S. hominis subsp. novobiosepticus was identified as homologue A, commonly found in S. aureus and many coagulase-negative staphylococcal species. The type strain of S. hominis subsp. novobiosepticus is ATCC 700236T. Descriptions of S. hominis subsp. novobiosepticus subsp. nov. and S. hominis subsp. hominis are given and the description of S. hominis is emended.

Phylogenetic analysis of 20 Pseudomonas strains (Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas chlororaphis) was conducted by using the nucleotide sequences of the genes for 16S rRNA, DNA gyrase B subunit (gyrB) and RNA polymerase σ70factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments. On the basis of gyrB and rpoD sequences, these strains were split into two major clusters: one including the type strain of P. putida and all biovar A strains and the other including all P. putida biovar B strains, P. fluorescens strains and the P. chlororaphis strain. In the phylogenetic tree reconstructed from the 16S rRNA sequences including variable regions, P. putida biovar A and B strains were not separated into two independent clusters, whereas in the phylogenetic tree reconstructed from the 16S rRNA sequences excluding the variable region sequences, these strains were separated into P. putida biovar A and biovar B clusters. The pairwise distances estimated from the variable regions of 16S rRNA correlated poorly with the synonymous distances estimated from the gyrB and rpoD genes. On the other hand, a highly significant correlation was observed between the pairwise distances estimated from the non-variable regions of 16S rRNA and the synonymous distances from gyrB and rpoD genes. Consequently, only the 16S rRNA sequences in the non-variable regions should be used for the phylogenetic analysis. The gyrB and rpoD analyses showed the necessity for the reclassification of P. putida biovar B strains.