- Volume 43, Issue 1, 1993

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16S rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus Aeromonas of the family Aeromonadaceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya ). The sequences were alligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Vibrio marinus clearly was on a line of descent that was remote from other vibrios. As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromonadaceae are. Also, Vibrio cholerae strains formed a separate group with Vibrio mimicus at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus Listonella was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas nautica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16S rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.

Studies were carried out to determine the underlying reasons for the phenotypic heterogeneity of Bacillus brevis Migula 1900. DNA reassociation measurements demonstrated that 31 of 37 strains segregated into seven groups of genetically related organisms. The members of the seven groups were not related genetically to selected presently recognized Bacillus species that are strictly aerobic and form oval spores. The results of the DNA relatedness studies and phenotypic comparisons suggested that two of these groups have not been described previously and that the five other groups probably represent previously, but inadequately, described species. Two of the five groups are proposed as new species and are given the revived names Bacillus agri and Bacillus centrosporus. Thus, the phenotypic heterogeneity of B. brevis sensu lato results from the inclusion of genetically unrelated organisms in the taxon and not from the inherent variability of genetically related strains.

Twenty-three strains of the seven recognized Listeria species were studied by using DNA-DNA optical hybridization. The level of error in the data was low. Our results supported the results of Rocourt et al. (J. Rocourt, F. Grimont, P. A. D. Grimont, and H. P. R. Seeliger, Curr. Microbiol. 7:383-388, 1982), although there was some overlap between Listeria monocytogenes and Listeria innocua. We suggest that there may be more than one cluster in the species L. monocytogenes or the species may form a large spectrum of relatedness. The level of intraspecies homology in L. monocytogenes is very broad, as determined in both this study and other studies.

Mycoplasmas isolated from the throats of a rhesus monkey and a baboon within 3 days of their arrival from India were shown to be serologically distinct from 104 previously recognized Mycoplasma and Acholeplasma spp. Two mycoplasma colonies were cloned and examined in detail for morphology, growth, and biochemical characteristics. The two strains were closely related and had the following properties: Guanine-plus-cytosine content of 32 mol %, requirement for sterol, arginine hydrolysis, and anaerobic growth. Glucose was not metabolized, and urea was not hydrolyzed. Strain 3T (= NCTC 11728) is the type strain of a new species, Mycoplasma indiense.

Two thermophilic, anaerobic, xylan-degrading bacteria, strains B6A-RIT (T = type strain) and LX-11T, were isolated from Frying Pan Springs in Yellowstone National Park. These organisms grew chemoorganotrophically by utilizing xylan and starch but not cellulose, as well as a number of di- and monosaccharides, including glucose and xylose. Both organisms had the same optimum temperature and pH for growth (60°C and pH 6.0). The fermentation products included acetate, ethanol, lactate, CO2, and H2. Both organisms were rod shaped and deposited sulfur on their cells. The major difference between the two isolates was in spore formation; strain LX-11T sporulated, whereas strain B6A-RIT were compared with other thermophilic, anaerobic, xylan-degrading bacteria by performing DNA-DNA hybridizations and total protein analyses in order to determine the relationships of these organisms. Three different groups were identified, and new taxonomic assignments are proposed. Clostridium thermocellum LQRI was least closely related to the other seven strains studied and is placed in group I, retaining its original taxonomic assignment. Clostridium thermosulfurogenes 4BT and new isolates B6A-RIT are closely related and fall into group II, for which the new genus Thermoanaerobacterium is proposed. Isolate LX-11T is designated Thermoanaerobacterium xylanolyticum sp. nov., and isolate B6A-RIT is designated Thermoanaerobacterium saccharolyticum sp. nov. Thermoanaerobacterium thermosulfurigenes 4BT (originally C. thermosulfurogenes ) is the type strain of the type species of the genus. Group III strains are placed in the genus Thermoanaerobacter ; this group includes Thermoanaerobacter ethanolicus JW200T, Clostridium thermohydrosulfuricum 39E and E100-69T, and Thermoanaerobium brockii HTD4T. Thermoanaerobium brockii HTD4T is renamed Thermoanaerobacter thermohydrosulfuricus comb. nov. C. thermohydrosulfuricum 39E is nearly identical to Thermoanaerobacter ethanolicus JW200T, and these organisms are considered members of the same species. Therefore, C. thermohydrosulfuricum 39E is renamed Thermoanaerobacter ethanolicus 39E; strain JW200 is the type strain of Thermoanaerobacter ethanolicus.

A new aerobic, gram-positive, motile coccus isolated from soil is described. Strain RA 333T (T = type strain) has the following characteristics: Menaquinone MK-9(H2); G+C content of DNA of 73.9 mol%; meso -diaminopimelic acid, glutamic acid, and alanine in a molar ratio of ca. 1:1:2 (type A1γ); and arabinose and galactose in the cell wall. The two sugars are contained in the cell wall as an arabinogalactan polymer. Mycolic acids are not present. The taxonomic characteristics of this organism are different from those of previously described aerobic, gram-positive cocci. The name Kineococcus aurantiacus gen. nov., sp. nov., is proposed. The type strain is strain RA 333, which has been deposited in the Institute for Fermentation, Osaka, as strain IFO 15268.

We analyzed the ribosomal AT-L30 proteins from 13 type strains of species belonging to the genera Microbispora and Actinomadura. The electrophoretic mobilities of the AT-L30 preparations from Microbispora strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that the members of the genus Microbispora are phylogenetically homogeneous. The results of partial amino acid sequencing of AT-L30 preparations from several representative Microbispora strains supported the separation of the genus Microbispora from other related genera. The amino acid sequences of the AT-L30 proteins from strains of species belonging to the genus Actinomadura sensu stricto displayed a diversity that exemplified the low levels of amino acid sequence homology within the genus. This diversity was considered to be a characteristic typical of the genus Actinomadura.

We describe the bacteriolytic activity of 377 group D Enterococcus isolates expressed towards 25 Enterococcus strains belonging to different species and Micrococcus luteus ATCC 4698. Of the 26 indicator strains used to reveal bacteriolytic activity, 5 were lysed by all of the strains of some species and were not lysed by all of the strains of other species. The use of these indicator strains allowed us to devise a new method to differentiate group D Enterococcus strains, based on qualitative analysis (lysis or no lysis of the indicator strains) of bacteriolytic activity. The bacteriolytic patterns obtained fell into six bacteriolytic groups corresponding (98% agreement) to species or groups of enterococci as determined by a comparison with data from a phenetic similarity study.

A two-dimensional electrophoresis method was devised for comparing ribosomal proteins from different bacteria. First-dimension separation of the proteins was carried out by nonequilibrium pH gradient gel electrophoresis in glass capillary tubes. Next, the first-dimension gels were developed in a slab gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Within about 3 h (total running time), 40 to 48 distinct and compact ribosomal protein spots were detected in each of the 34 strains belonging to eight genera of gram-positive bacteria which we studied. Similarity coefficients (SAB values) among the proteins of Corynebacterium and Arthrobacter strains were calculated by determining the numbers of coincident spots obtained by coelectrophoresis. The SAB values between strains having DNA relatedness values of more than 61% (strains of the same species) were greater than 0.85, whereas the SAB values between strains of different species were less than 0.45. Ribosomal protein analysis by our improved method is a potential tool for species identification of bacteria.

Phenotypic data and phospholipid ester-linked fatty acid profiles indicate that pigmented bacterial strains isolated from a hypersaline Antarctic lake are members of the “flavobacterium-bacteroides” phylum and may represent new taxa. Nearly complete 16S rRNA sequences were obtained for representative strains by directly sequencing the polymerase chain reaction-amplified 16S rRNA gene. Sequence signatures confirmed that these organisms were members of the flavobacterium-bacteroides phylum. A phylogenetic analysis, in which the sequences of the Antarctic strains were compared with a large number of sequences available for members of the flavobacterium-bacteroides phylum, showed that the Antarctic strains were phylogenetically distinct. The new species cluster with a group of organisms that contains the type species of the genus Flavobacterium, Flavobacterium aquatile. Two new species are described, for which the names Flavobacterium gondwanense and Flavobacterium salegens are proposed; strains ACAM 44 (= DSM 5423) and ACAM 48 (= DSM 5424) are the type strains of F. gondwanense and F. salegens, respectively.

Isolate PCM 2404T (T = type strain) from the Polish Collection of Microorganisms is proposed as the type strain of a new species, Streptomyces spitsbergensis. This new species is characterized by a nonfragmenting vegetative mycelium, rectiflexibiles spore chains, a type I cell wall, type PII phospholipids, a lack of mycolic acids and major glycolipids, and a G+C content of 71 mol%. A comparison with related species of the genus confirmed the validity of the proposed taxon.

Clustering and ordination were used to analyze the results of a study involving phenotypic characterization (61 tests) of 197 strains of gram-negative, oxidase-positive, rod-shaped bacteria isolated from carcharhinid sharks. Also included in the data set were data for 39 reference strains representing the genera Aeromonas, Plesiomonas, and Vibrio. Unweighted pair group mathematical averaging cluster analysis resolved nine major groups of operational taxonomic units (OTUs) at simple matching coefficient (SSM) values of ≥66.5%. In turn, each group contained one or more OTU clusters (phena) at SSM values of ≥72.6%. A total of 27 phena were resolved; 14 phena were identified as Vibrio species and contained 182 of the 236 OTUs in the study. Additional phena were identified as members of the genus Aeromonas (two phena), the genus Photobacterium (three phena), the genus Alteromonas (two phena), and Plesiomonas shigelloides (one phenon). One phenon resembled descriptions of the genera Moraxella and Neisseria and Pasturella piscicida. The remaining four phena were not identified. The largest phena were identified as Vibrio damsela (52 OTUs) and Vibrio alginolyticus (48 OTUs). Vibrio carchariae and Vibrio harveyi formed subclusters (SSM values, ≥82%) within the third largest phenon (24 OTUs), which was resolved at an SSM value of ≥72.6%. Human pathogens (e.g., Vibrio cholerae ) and fish pathogens (e.g., Aeromonas salmonicida, Pasteurella piscicida, and V. damsela were isolated from the sharks. Approximately 58% of all of the strains isolated and tested in this study were urease positive.

Four strains of a novel Helicobacter species were isolated from the stomachs of cheetahs (Acinonyx jubilatus ) with gastritis. These isolates were phenotypically similar to Helicobacter pylori. The isolates were gram-negative, spiral bacteria which grew under microaerophilic conditions at 37°C, but not at 25 or 42°C, and produced urease, catalase, oxidase, alkaline phosphatase, and gamma-glutamyl transpeptidase. The isolates did not ferment glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, or arabinose; hydrolyze hippurate or indoxyl acetate; or reduce nitrate. They did not produce H2S from triple sugar iron agar, and they did not grow in the presence of 1.0% glycine or 1.5% NaCl. They were resistant to nalidixic acid and sensitive to cephalothin and metronidazole. Cells were typically 0.3 by 2.0 μm and possessed tufts of two to five sheathed, monopolar flagella. The G+C content of strain 90-119 was 30 mol%. Cluster analysis of densitometry scans of polyacrylamide protein gels revealed more than 70% similarity of the cheetah isolates to H. pylori, less than 60% similarity to Helicobacter fells, and less than 50% similarity to Helicobacter mustelae. Complete 16S rRNA sequences were determined for two of the cheetah isolates. Phylogenetic analysis was performed by comparing the cheetah sequences to those of 19 reference strains, inluding H. pylori, H. felis (two strains), H. mustelae, Helicobacter muridarum, “Flexispira rappini,” Wolinella succinogenes, Campylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter sputorum subsp. bubulus, a Campylobacter sp. (pig isolate), [Bacteroides] gracilis, and [Bacteroides] ureofyticus. The 16S rRNA sequences for 13 of the 19 reference species have not previously been reported. Phylogenetic analysis demonstrated that the cheetah isolates were most closely related to H. pylori (97.4% similarity), H. felis (96.1% similarity), and H. mustelae (93.4% similarity). On the basis of these findings, we propose that these isolates represents a novel species of Helicobacter, which we designate Helicobacter acinonyx. The type strain is 90-119 (CCUG 29263, ATCC 51101).

In previous studies, gram-positive bacteria which grew rapidly with peptides or an amino acid as the sole energy source were isolated from bovine rumina. Three isolates, strains C, FT (T = type strain), and SR, were considered to be ecologically important since they produced up to 20-fold more ammonia than other ammonia-producing ruminal bacteria. On the basis of phenotypic criteria, the taxonomic position of these new isolates was uncertain. In this study, the 16S rRNA sequences of these isolates and related bacteria were determined to establish the phylogenetic positions of theorganisms. The sequences of strains C, FT, and SR and reference strains of Peptostreptococcus anaerobius, Clostridium sticklandii, Clostridium coccoides, Clostridium aminovalericum, Acetomaculum ruminis, Clostridium leptum, Clostridium lituseburense, Clostridium acidiurici, and Clostridium barkeri were determined by using a modified Sanger dideoxy chain termination method. Strain C, a large coccus purported to belong to the genus Peptostreptococcus, was closely related to P. anaerobius, with a level of sequence similarity of 99.6%. Strain SR, a heat-resistant, short, rod-shaped organism, was closely related to C. sticklandii, with a level of sequence similarity of 99.9%. However, strain FT, a heat-resistant, pleomorphic, rod-shaped organism, was only distantly related to some clostridial species and P. anaerobius. On the basis of the sequence data, it was clear that strain FT warranted designation as a separate species. The closest known relative of strain FT was C. coccoides (level of similarity, only 90.6%). Additional strains that are phenotypically similar to strain FT were isolated in this study. On the basis of phenotypic and phylogenetic differences, we believe that strain FT represents a new species of the genus Clostridium, for which we propose the name Clostridium aminophilum.

The protein patterns of whole cells of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum were studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein patterns of the 16 strains of E. rhusiopathiae and E. tonsillarum studied, including the type strains of these two species, resembled each other, except that there were 71-, 41-, 34-, and 26-kDa proteins in the E. rhusiopathiae pattern and 74-, 44-, 36-, and 25-kDa proteins in the E. tonsillarum pattern. This observation indicates that there is some phenotypic heterogeneity in the genus Erysipelothrix. In addition, the protein patterns of E. rhusiopathiae serotype reference strains representing serotypes 1 through 23 and type N were compared. The protein patterns of serotype 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 19, and 21 and type N strains were similar to the pattern of the type strain of E. rhusiopathiae (strain ATCC 19414). Conversely, the protein patterns of serotype 3, 7, 10, 14, and 20 strains were very similar to the pattern of the type strain of E. tonsillarum (strain ATCC 43339). An atypical pattern was observed in serotype 13, 17, 18, 22, and 23 strains. These results suggest that this method may be used as an aid in studying the taxonomy of these bacteria.

Pseudomonas mixta (type strain, ACM 1762 [=ATCC 49108], an actively dextranolytic species that possesses both lateral and polar flagella, was compared with the strictly aerobic, rod-shaped, chitinolytic bacterium “Pseudomonas chitinolytica„ ACM 3522T (= CNCM I-804) (T = type strain), which has a similar flagellation pattern, by performing phenotypic characterization and DNA-DNA hybridization studies and by analyzing DNA base compositions and 16S rRNA sequences. Our results indicated that “P. chitinolytica„ ACM 3522T was phenotypically and genotypically distinct from P. mixta and other phenotypically analogous Pseudomonas spp., Xanthomonas maltophilia, and other aerobic chitin degraders. The 16S rRNA sequences of strains ACM 1762T and ACM 3522T were found to be very similar (97%) to each other and indicated that these organisms are proteobacteria that belong to the β subclass. The strains were deeply branched in the β subclass and were distinct from other pseudomonads, including Pseudomonas cepacia, and from Comamonas testosteroni. On the basis of phenotypic, genotypic, and phylogenetic evidence, it is proposed that P. mixta and “P. chitinolytica„ ACM 3522T represent two distinct species in a new genus called Telluria. Thus, the genus Telluria gen. nov. contains Telluria mixta comb. nov. and Telluria chitinolytica sp. nov., which are strictly aerobic, rod-shaped, soil-dwelling bacteria that are active polysaccharide degraders.

Four strains of orange- or red-pigmented bacteria isolated from freshwater surfaces were shown to synthesize bacteriochlorophyll under aerobic conditions. These strains shared unusual morphological features, such as acellular stalks, crateriformlike structures, and buds, with bacteria in the order Planctomycetales. However, comparisons of 16S rRNA sequences showed them to be members of the α-4 subdivision of the class Proteobacteria and most closely related to the marine aerobic bacteriochlorophyll-synthesizing bacterium Erythrobacter longus. They also differ from members of the Planctomycetales phenotypically in their synthesis of bacteriochlorophyll and possession of a peptidoglycan cell wall. They can be distinguished from E. longus on the basis of their 16S rRNA sequence, the G+C content of their DNA, cellular fatty acid composition, and carbon substrate spectrum. A new genus, Porphyrobacter, with a single species, P. neustonensis gen. nov., sp. nov., is proposed for these strains. The type strain is ACM 2844.

A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a singlepolar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16S rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Proteobacteria. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudomonas and allowed placement in the beta subclass of the Proteobacteria. Within the beta subclass, strain AXT is related to Iodobacter fluvatile. The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Iodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis.

A new genus, Rathayibacter, is proposed to accommodate three species of gram-positive, aerobic, coryneform bacteria previously placed in the genus Clavibacter (Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., and Rathayibacter iranicus comb. nov.), as well as six strains that were isolated from annual cereal grasses, may be responsible for ryegrass toxicity, and are very similar to the recently described organism Clavibacter toxicus sp. nov. (I. T. Riley and K. M. Ophel, Int. J. Syst. Bacteriol. 42:64-68, 1992). The properties of members of the genus Rathayibacter include coryneform morphology, peptidoglycan based on 2,4-diaminobutyric acid (type B2γ), predominant menaquinones of the MK-10 type, and phosphatidylglycerol and diphosphatidylglycerol as basic polar lipids. The DNA base compositions range from 63 to 72 mol% G+C. The members of the new genus form a phenetic cluster distinct from Clavibacter spp. at a level of 71% (simple matching coefficient) and exhibit 7 to 9% DNA-DNA reassociation with strains of Clavibacter spp. In contrast to Clavibacter spp., most Rathayibacter strains are associated with nematodes belonging to the genus Anguina. The Rathayibacter species differ from species belonging to related genera (e.g., Clavibacter and Agromyces species) in the following characteristics: Menaquinone and whole-cell sugar compositions, results of lysozyme-sodium dodecyl sulfate test (which indicates differences in cell wall composition), ability to utilize a number carbon sources, resistance to bacteriocins of some Clavibacter spp., and other characteristics. The Rathayibacter species can be differentiated from each other by following characteristics: Presence or absence of xylose and galactose in the cell walls, fatty acid composition, ability to assimilate various sources of carbon and nitrogen, hydrolytic activity, tolerance to 5% NaCI and 0.03% potassium tellurite, susceptibility to iranicin, and absence or presence of plasmids of certain sizes. The type species of the genus Rathayibacter is R. rathayi. The type strains of the species are R. rathayi VKM Ac-1601 (= ICMP 2574), R. iranicus VKM Ac-1602 (= ICMP 3496), and R. tritici VKM Ac-1603 (= ICMP 2626).