- Volume 44, Issue 1, 1994

Phylogenetic relationships among species of the genus Rhodobacter and related taxa were elucidated by studying 16S rRNA sequence information and genomic DNA homology data. The 16S rRNA gene was amplified by the PCR and was sequenced directly by a combined method consisting of cycle sequencing and automated fluorescence detection. Pairwise sequence comparisons and a distance matrix analysis showed that the Rhodobacter species could be divided into two major clusters; one cluster included the freshwater and terrestrial species, and the other cluster contained the marine species. The cluster containing the freshwater Rhodobacter species also included Rhodopseudomonas blastica and was linked more closely to the chemotroph Paracoccus denitrificans and the aerobic phototroph Roseobacter denitrificans than to the cluster containing the marine Rhodobacter species. Genomic DNA-DNA hybridization data supported the results of 16S ribosomal DNA sequence comparisons. With few exceptions, the marine Rhodobacter species can be differentiated phenotypically from the freshwater species on the basis of salt requirement for optimal growth, sulfide tolerance, final oxidation product of sulfide, and polar lipid composition. Thus, we propose that all marine Rhodobacter species should be transferred to the genus Rhodovulum gen. nov.; Rhodovulum sulfidophilum comb. nov. is the type species of this genus.

The phylogenetic position and various genotypic, chemotaxonomic, and classical phenotypic characteristics of 21 gram-negative avian isolates were studied. These strains constitute a genotypically homogeneous taxon in rRNA superfamily V, as shown by DNA-rRNA hybridization data. Determination of the 16S rRNA sequence of this taxon revealed its detailed position within the “flavobacter” subgroup of the “flavobacter-bacteroides” phylum as described by Gherna and Woese (R. Gherna and C. R. Woese, Syst. Appl. Microbiol. 15:513-521, 1992). This new taxon is only distantly related to other members of the “flavobacter-bacteroides” phylum and is therefore given separate generic status. The DNA-DNA binding values for members of this taxon, for which we propose the name Ornithobacterium rhinotracheale, confirmed that all of the strains are highly interrelated (DNA-DNA binding values greater than 90% were measured). The G+C contents of members of this taxon are between 37 and 39 mol%. An analysis of the cellular proteins and fatty acids and classical phenotypic characteristics allowed us to distinguish O. rhinotracheale from phenotypically similar taxa, such as Riemerella anatipestifer and Capnocytophaga species. The respiratory quinone content (menaquinone 7) and carbohydrate pattern of O. rhinotracheale conform with the respiratory quinone contents and carbohydrate patterns of other members of rRNA superfamily V.

The 16S ribosomal DNAs (rDNAs) of two strains of Francisella tularensis and one strain of Francisella philomiragia were sequenced. On the basis of phylogenetic analysis data, the genus Francisella was placed in the ? subclass of the Proteobacteria. The most closely related organism was the intracellular bacterium Wolbachia persica. The sequenced 16S rDNA molecules of the Francisella species exhibited very high levels of similarity (98.5 to 99.9%). Two variable regions, comprising 390 to 450 nucleotides of the 16S rDNA molecules of 17 additional Francisella strains, including members of the species F. tularensis and F. philomiragia, were also sequenced. At most, six nucleotide differences were observed among the sequences of the F. tularensis strains. The sequence of Francisella novicida was virtually identical to the sequences of the F. tularensis strains, thereby supporting the hypothesis that these organisms are members of the same species. On the basis of the observed differences, primer pairs were designed to distinguish strains by using the PCR at the genus, species, and subspecies levels. This permitted sensitive identification of strains belonging to the genus Francisella and discrimination of the species F. tularensis and F. philomiragia.

Two genetically diverse groups of strains were identified among cultures of Xanthomonas campestris pv. vesicatoria isolated from plants with bacterial spot of pepper and tomato. Group A strains do not pit pectate gels or hydrolyze starch, whereas group B strains are strongly positive for these reactions. Group A strains cause a hypersensitive reaction in plants of tomato breeding line Hawaii 7998, but group B strains do not. Other differences between the two groups of strains were discovered in tests for utilization of carbon compounds, serology, fatty acid profiles, silver-stained protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and DNA restriction enzyme digestion profiles. The levels of DNA homology between strains belonging to the same group were more than 74%, but the levels of DNA homology between strains belonging to different groups were less than 46%. The two groups of strains have different genetic backgrounds, but cause essentially the same disease of tomato and pepper.

A genome and fatty acid analysis of 16 Pseudomonas stutzeri reference strains having DNA compositions ranging from 62.2 to 65.5 mol% G+C was performed by pulsed-field gel electrophoresis of Xba I and Spe I macrorestriction fragments and gas chromatography of total cellular fatty acids. Macrorestriction fragment patterns were evaluated by using previously described algorithms (D. Grothues and B. Tümmler, Mol. Microbiol. 5:2763-2776, 1991), and the results allowed us to subdivide the species into two groups which correlated with G+C content. Two examples of recent strain divergence were observed among clinical isolates, but in general a marked degree of heterogeneity was observed in the macrorestriction fragment patterns, and even phenotypically similar strains produced divergent patterns. While the differences were not sufficiently great to exclude any strain from P. stutzeri, they suggest that recombination and niche-specific selection may be significant factors responsible for generating and maintaining the heterogeneity inherent in the species. Genome sizes were estimated from the sums of Spe I restriction fragment sizes and ranged from 3.4 to 4.3 Mbp; the genome sizes of the low-G+C-content strains (G+C contents, approximately 62 mol%) were confined to a narrow range between 3.9 and 4.1 Mbp. An examination of the distributions of macrorestriction fragments resulting from digestion with Xba I and Spe I showed that both distributions differed significantly from the expected (random) distribution, suggesting that there is a supragenic level of chromosomal organization. An analysis of fatty acid methyl ester data by using Microbial Identification System software revealed a similar correlation between phenotype and G+C content, indicating that division of the species is possible by the method used in this study. For comparative purposes, a numerical analysis of previously reported substrate utilization data (N. J. Palleroni, M. Doudoroff, R. Y. Stanier, R. E. Solánes, and M. Mandel, J. Gen. Microbiol. 60:215-231, 1970) was performed. The results of this analysis revealed that there was a relationship among strains which showed no correlation with the results obtained from either the macrorestriction fragment analysis or the fatty acid methyl ester analysis.

Fifty-two strains of photosynthetic rhizobia isolated from stem and root nodules of Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene schemperi, and Aeschynomene sensitiva were compared with reference rhizobial strains representing Rhizobium, Bradyrhizobium, and Azorhizobium species by performing a multivariate statistical analysis of 150 phenotypic characteristics. Eight phena, some of which contained subphena, were identified. Aeschynomene rhizobia which synthesize bacteriochlorophyll were grouped in phenon 6, which contained three subphena, while nonphotosynthetic Aeschynomene rhizobia were grouped in phena 1 and 2. Our results indicate that the photosynthetic rhizobia nodulating Aeschynomene species belong to a phenon (phenon 6) that is separate from the genera Bradyrhizobium (phena 1 to 5), Azorhizobium (phenon 7), and Rhizobium (phenon 8).

The 16S rRNA gene sequences of “Bacillus aminovorans” and several species considered to be phylogenetically related to the group 2 bacilli of Ash et al. (C. Ash, J. A. E. Farrow, S. Wallbanks, and M. D. Collins, Lett. Appl. Microbiol. 13:202–206, 1991) were determined. A comparative analysis of the sequence data revealed that the round-spore-forming group 2 bacilli, together with some asporogenous taxa (the genera Caryophanon, Exiguobacterium, Kurthia, Planococcus), form a phylogenetically distinct cluster that is only remotely related to Bacillus subtilis, the type species of the genus Bacillus. Within this cluster, planococci, kurthiae, Caryophanon spp., and two lines defined by Bacillus sphaericus and Bacillus fusiformis and by Sporosarcina ureae, Bacillus pasteurii, Bacillus globisporus, and Bacillus psychrophilus were found to be distinct genera. Exiguobacterium aurantiacum and Brevibacterium acetylicum were found to form a distinct clade, which was peripherally related to this cluster. “B. aminovorans” exhibited no specific relationship with the group 2 bacilli or with any of the other reference species examined.

The phylogeny of bacteria belonging to the genus Nitrobacter was investigated by sequencing the whole 16S rRNA gene. The average level of similarity for the three Nitrobacter strains examined was high (99.2%), and the similarity level between Nitrobacter winogradskyi and Nitrobacter sp. strain LL, which represent two different genomic species, was even higher (99.6%). When all of the Nitrobacter strains and their phylogenetic neighbors Bradyrhizobium and Rhodopseudomonas species were considered, the average similarity level was 98.1%. When complete sequences were used, Nitrobacter hamburgensis clustered with the two other Nitrobacter strains, while this was not the case when partial sequences were used. The two Rhodopseudomonas palustris strains examined exhibited a low similarity level (97.6%) and were not clustered.

Pectinophiles are bacteria that utilize pectin and only a few related compounds as substrates. Obligately anaerobic pectinophiles have been isolated from the intestinal tracts and gingivae of humans and from the rumina of cattle. We isolated three strains of pectinophilic bacteria from colonic contents of pigs but were unable to isolate pectinophiles from the rumen contents of four sheep, even when the animals were fed a high-pectin diet. The pectinophiles isolated from pigs were strictly anaerobic, motile, gram-positive rods (0.36 to 0.56 by 2.4 to 3.1 μm). Pectin, polygalacturonic acid, and gluconate were the only substrates that supported rapid growth. All three strains grew slowly on either lactose or cellobiose and fermented fructose after a lag of several days. Pectin was degraded by means of an extracellular pectin methylesterase and a Ca2+-dependent exopectate lyase. A comparison of the 16S rRNA sequences of these isolates with the 16S rRNA sequences of other gram-positive bacteria revealed a specific relationship with Lachnospira multipara (level of similarity, 94%). The Gram reaction, formation of spore-like structures, and the utilization of lactose and cellobiose differentiated the pig isolates from previously described pectinophiles. The pig isolates represent a previously undescribed species of the genus Lachnospira, for which we propose the name Lachnospira pectinoschiza.

Organisms with the typical characteristics of mycoplasmas were isolated from the tracheae of buzzards, saker falcons, and griffon vultures. All isolates obtained from the same bird species appeared to be serologically identical and distinct from the isolates obtained from the other two bird species and from the previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that the isolates belong to three new species, for which the names Mycoplasma buteonis (seven strains obtained from buzzards), Mycoplasma falconis (six strains obtained from saker falcons), and Mycoplasma gypis (eight strains obtained from griffon vultures) are proposed. M. buteonis ferments glucose, does not hydrolyze arginine or urea, reduces tetrazolium chloride and potassium tellurite anaerobically, does not produce films and spots, and does not possess phosphatase activity. M. falconis does not ferment glucose, hydrolyzes arginine but not urea, reduces tetrazolium chloride and potassium tellurite anaerobically, does not produce films and spots, and does not possess phosphatase activity. M. gypis does not ferment glucose, hydrolyzes arginine but not urea, reduces tetrazolium chloride and potassium tellurite aerobically and anaerobically, produces films and spots, and possesses phosphatase activity. The three new species lyse avian, bovine, equine, human, ovine, porcine, rabbit, and guinea pig erythrocytes, but do not adsorb these erythrocytes. The type strain of M. buteonis is Bb/T2g (= ATCC 51371), the type strain of M. falconis is H/T1 (= ATCC 51372), and the type strain of M. gypis is B1/T1 (= ATCC 51370).

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the “Bacillus cereus group” of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

A toluene-degrading bacterial strain isolated from bioreactors was identified as a member of the genus Rhodococcus on the basis of the following characteristics: mero-diaminopimelic acid, arabinose, galactose, and glucose are the diagnostic cellular sugars; the mycolic acids contain 33 to 36 carbon atoms; and the formation of a branching mycelium is followed by marked fragmentation of the mycelium into irregular rod-shaped to coccoid units. DNA-DNA hybridization analyses performed with type strains of Rhodococcus species showed that this strain is less than 70% related to other species that have similar phenotypic characteristics. On the basis of these findings, we propose that this strain should be described as a new species, Rhodococcus zopfii, in honor of Wilhelm Friedrich Zopf. The type strain is strain T1.

An anaerobic and thermophilic alkaliphile, strain JW/YL23-2T (T = type strain), was isolated from sewage sludge obtained from a sewage plant in Atlanta, Ga. At pH 10.1 and 50°C, the doubling time of this strain was 19 min. Strain JW/YL23-2T, a motile rod-shaped bacterium with 2 to 12 peritrichous flagella, exhibited a negative Gram stain reaction but was gram-type positive as judged by the polymyxin B test. No heat-stable (85°C, 15 min) endospores were detected. At 50°C, growth occurred at pH values ranging from 7.0 to 11.0; the optimum pH was 9.6 to 10.1. The temperature range for growth ranged from 27 to 57.5°C; the optimum temperature was 48 to 51°C (pH 10.1). Dissimilatory sulfate reduction was not detected. The organism utilized glucose, fructose, sucrose, maltose, cellobiose, and Casamino Acids. The DNA G+C content was 32 mol% (as determined by chemical analysis). A16S rRNA sequence analysis revealed a 2% inferred evolutionary distance to Clostridium paradoxum. However, the cell wall type of strain JW/YL23-2T was A4β (L-Orn-D-Asp), while that of C. paradoxum was A1τ (m-diaminopimelic acid direct). On the basis of the alkaline pH values and high temperatures for optimal growth, the inability to form spores, and other characteristics different from C. paradoxum characteristics, strain JW/YL-23-2 was placed in a new species, Clostridium thermoalcaliphilum; JW/YL23-2 (= DSM 7309) is the type strain of this new species.

A gene for a ribosomal protein, rps3, was amplified by PCR and sequenced from representatives of the class Mollicutes. Alignments of the deduced amino acid sequences allowed the construction of a phylogeny that is consistent with the phylogenetic trees created from 5S and 16S rRNA comparisons, including the position of the former Acholeplasma florum on the Mycoplasma branch, rather than with the classical Acholeplasmataceae. Additional confirmation of the phylogeny comes from the deduction that the UGA triplet encodes tryptophan in the rps3 gene from Mesoplasma florum, as it does in the mycoplasmas and spiroplasmas. The sequence data from Acholeplasma axanthum 743 and Acholeplasma sp. strain J233 allow refinements to the phylogenetic tree within the Acholeplasmataceae, providing evidence that the sterol requirement of Anaeroplasma abactoclasticum (order Anaeroplasmatales) is a derived trait. It was also evident that the nonhelical plant-pathogenic members of the class Mollicutes, referred to as mycoplasmalike organisms or phytoplasmas, are more closely related to the true acholeplasmas (Acholeplasma laidlawii and strain J233) than to other members of the Mollicutes.

The genetic relationships of Bacillus cereus and of the Bacillus thuringiensis serovars were assessed from measurements of DNA reassociation. A study of 8 to 10 strains each of 13 of the most commonly encountered serovars revealed that the levels of intragroup DNA relatedness for most serovars ranged from 90 to 100%. In contrast, B. thuringiensis serovars canadensis and kenyae consisted of two DNA relatedness groups, each of which exhibited levels of intragroup relatedness of 80% or higher and levels of intergroup relatedness of 60 to 70%. Analyses of DNA relatedness performed with all of the serovars revealed that the taxa were segregated into 11 phena differentiated from each other at about the 65% level; within each phenon the level of relatedness was 80% or higher. Three phena contained strains belonging to more than one serovar; B. thuringiensis serovars alesti and dendrolimus clustered in phenon 1, serovars aizawai, kurstaki, galleriae, and morrisoni clustered in phenon 7, and serovar darmstadiensis and some strains of serovar kenyae clustered in phenon 11. The levels of DNA relatedness between B. cereus and B. thuringiensis strains ranged between 65 and 70%. My results suggest that many of the B. thuringiensis serovars are genetically distinct but closely related.

The 16S ribosomal DNA sequences of representative members of the family Peptococcaceae were determined. The members of the family examined were divided into the following four phylogenetic groups: Peptococcus niger ATCC 27731T (T = type strain), the Sarcina-Peptostreptococcus anaerobius group, the ruminococcus-coprococcus group, and the peptostreptococcus group. Peptococcus niger, the type species of the family, was not related to other members of the family. Peptostreptococcus anaerobius ATCC 27337T, the type strain of the type species of the genus Peptostreptococcus, was closely related to Clostridium sordellii NCIB 10717T (level of sequence similarity, 85%). Sarcina ventriculi GIFU 7886, a spore-forming anaerobic gram-positive coccus, clustered with Clostridium perfringens ATCC 13124T at a similarity value of 91%. Members of the Sarcina-Peptostreptococcus anaerobius group clustered with clostridia at similarity values ranging from 85 to 91%. The type strains of Peptostreptococcus prevotii, Peptostreptococcus asaccharolyticus, Peptostreptococcus micros, and Peptostreptococcus magnus clustered at levels of sequence homology of 84 to 93%. This cluster was not included in the Peptococcus niger group or the Peptostreptococcus anaerobius group. Thus, these members of the genus Peptostreptococcus should be separated from the other members of the genus and also from members of the family Peptococcaceae. The sequence of Peptostreptococcus productus ATCC 27340T was different from the sequences of Peptostreptococcus anaerobius and Peptococcus niger. The sequence of Streptococcus hansenii ATCC 27752T, a strictly anaerobic strain, was different from the sequences of other streptococci; this strain clustered with Peptostreptococcus productus, coprococci, and ruminococci. Several phenotypic characteristics of Streptococcus hansenii ATCC 27752T were similar to characteristics of ruminococci. These organisms require fermentable carbohydrates and do not produce butyric acid from glucose. Thus, we propose that Peptostreptococcus productus and Streptococcus hansenii should be transferred to the genus Ruminococcus.

A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen. The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.

The taxonomic position of “Bacillus aneurinolyticus” was determined by numerical analyses based on phenotypic characteristics and whole-cell proteins profile, chemosystematic data, DNA base composition, and DNA relatedness. “B. aneurinolyticus” strains were separated into “B. aneurinolyticus” and Bacillus migulanus by DNA relatedness. This result correlated well with the clusters obtained from numerical analyses based on phenotypic characteristics and whole-cell proteins profile. “B. aneurinolyticus” was clearly distinct from other Bacillus species phenotypically and genetically. We propose the revival of the name Bacillus aneurinolyticus.

A total of 63 strains of rhizobia isolated from Hainan Province, a tropical region of the People’s Republic of China, and 27 representative strains belonging to the genera Rhizobium, Brady rhizobium, and Agrobacterium were compared by performing numerical taxonomy, DNA hybridization, and DNA base composition analysis to determine the relationships among these rhizobia. The results indicated that the strains isolated from Hainan Province fell into two distinct phena, the slowly growing rhizobia and the fast-growing rhizobia. The slowly growing rhizobia, which formed three subphena that seemed to be three subspecies, are Bradyrhizobium japonicum strains. The fast-growing strains belong to the genus Rhizobium and might be further divided into three specific groups. Sometimes both slowly growing rhizobia and fast-growing rhizobia were isolated from host plants belonging to the same genus or species or even from the same nodule. There was no correlation between hosts and the distribution of rhizobia in the subphena. Isolates obtained from members of the same host genus or species fell into different groups or subgroups.