- Volume 40, Issue 4, 1990

Four strains of tetrodotoxin-producing bacteria isolated from a red alga and from pufferfish were characterized. Two of these strains are members of the genus Listonella MacDonell and Colwell. The phenotypic characteristics, guanine-plus-cytosine contents, and base sequences of the 16S rRNAs of these organisms indicated that they are members of Listonella pelagia (Vibrio pelagius) biovar II. The other two strains are members of the genus Alteromonas Baumann et al. and the genus Shewanella MacDonell and Colwell. These two strains are mutually distinct and distinct from the previously described Alteromonas and Shewanella species and therefore are placed in new species. The names Shewanella alga and Alteromonas tetraodonis are proposed for these organisms; the type strains are strains OK-1 and GFC, respectively.

The recent isolation and characterization of two marine strains of Flectobacillus marinus are significant for precise definition of this species, which previously was based on a single strain. Polyphasic taxonomic studies showed that this species is not closely related to the only other species, F. major, of the genus Flectobacillus. Because F. marinus cannot be assigned to any other genus, a new genus, Cyclobacterium, in the family Spirosomaceae is proposed. We recommend that F. marinus be transferred to the new genus, with Cyclobacterium marinus comb. nov. as the type species. The type strain is C. marinus Raj (= ATCC 25205). Descriptions of the new genus and its species, necessitated by this change, and a key for differentiation from other family members are presented together with an emended description of the genus Flectobacillus.

An extensive phenotypic description and an improved classification and nomenclature of the genus Xanthomonas are presented. A total of 266 strains obtained from different geographical areas, including representative strains of all species of the genus Xanthomonas and most pathovars of Xanthomonas campestris, as well as strains which might be genetically related to the genus Xanthomonas, were examined for 295 morphological, biochemical, and physiological features. Similarities among the strains were expressed numerically by using the coefficient of Sokal and Michener. Clustering was performed by using the unweighted average pair group method. The conclusions described below were reached. (i) The genus Xanthomonas comprises at least the following eight phena: X. campestris, Xanthomonas albilineans, Xanthomonas axonopodis, Xanthomonas fragariae, Xanthomonas populi, Xanthomonas maltophilia, Xanthomonas oryzae Swings et al. 1990, and X. campestris pv. graminis Egli and Schmidt 1982 [not X. campestris pv. graminis (Egli et al. 1975) ISPP List 1980]. (ii) X. populi (Ridé 1958) Ridé and Ridé 1978 is a separate species. (iii) X. maltophilia Swings et al. 1983 forms a separate species. (iv) X. campestris pv. oryzae ISPP List 1980 can no longer be regarded as pathovar of X. campestris, and its recent reclassification as a new species, X. oryzae (Swings et al., Int. J. Syst. Bacteriol. 40:309-311, 1990), is supported. (v) X. campestris pv. graminis Egli and Schmidt 1982 [not X. campestris pv. graminis (Egli et al. 1975) ISPP List 1980] seems to form a separate complex of highly related pathovars obtained from members of the Poaceae; the taxonomic implications of this are discussed. (vi) Strains of nearly all X. campestris pathovars cluster together in the X. campestris phenon. Within this species we were able to differentiate some entities on phenotypic grounds; these groups sometimes corresponded to named pathovars (e.g., X. campestris pv. manihotis, X. campestris pv. cassavae, X. campestris pv. phlei). In several other cases, pathovars were found to be heterogeneous. (vii) A number of dubious Pseudomonas species were identified as members of or as being close to Xanthomonas species. Both Pseudomonas betle and Pseudomonas hibiscicola are synonyms of X. maltophilia. We also confirmed that Pseudomonas mangiferaeindicae, Pseudomonas vitiswoodrowii, and Pseudomonas gardneri belong to X. campestris. (viii) Forty phenotypic features allow the differentiation of the eight Xanthomonas phena. (ix) A number of additional features of the genera Xanthomonas and Xylophilus are described.

DNA hybridization and extracellular-polysaccharide (EPS) analyses were performed for 19 strains of Butyrivibrio fibrisolvens and six strains of Butyrivibrio crossotus, Lachnospira maltipara, Roseburia cecicola, and Eubacterium rectale. Both methods showed that these strains constitute a heterogeneous group of bacteria, and both methods sorted the strains into related groups. As determined by DNA analysis, 20 strains formed five separate groups of closely related bacteria. Nine of these strains were closely related to two previously established groups of B. fibrisolvens. These two groups, group 1 and group 3, contain 11 and 8 strains, respectively, and could represent new species. The type strain of B. crossotus, an isolate from human feces, was closely related genetically to five of the strains presently identified as B. fibrisolvens, which were isolated from rumina of bison. The groups formed by EPS analysis were similar to those formed by DNA analysis. Our data suggest that for some groups of microorganisms EPS analysis may be a useful adjunct to DNA analysis to clarify complex taxonomic relationships.

Five strains of a gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rod-shaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from tomatoes (three strains), a banana, and a cucumber. All of the strains produced a water-soluble pink pigment. As determined by DNA hybridization (hydroxyapatite method) these five strains were 85 to 100% related in 60 and 75°C reactions, and related sequences exhibited 1% or less base sequence divergence, indicating that the organisms are members of a single species. These bacteria were most closely related to Erwinia rhapontici (68 to 72% at 60°C, 42 to 44% at 75°C, 10.5% divergence) and to hybridization group VIII in the Enterobacter agglomerans (Pantoea agglomerans, Erwinia herbicola) complex (64% at 60°C, 32% at 75°C, 14.5% divergence). Phenotypic differentiation from Erwinia rhapontici, which also produces a water-soluble pink pigment, is based on negative reactions by the new species in tests for methyl red, N-acetylglucosamine, dl-tartrate assimilation, and acid production from amygdalin, dulcitol, d-fucose, β-gentiobiose, α-methyl-d-glucoside, glycerol, d-lyxose, melezitose, d-turanose, xylitol, and d-xylose and a positive reaction for acetoin (Voges-Proskauer test). On the basis of these data, the name Erwinia persicinus is proposed for the new organism. The type strain is strain HK 204 (= AJ 2716 = CDC 9108-82 = IAM 12843 = JCM 3704 = ATCC 35998).

Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: The type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).

5S rRNA sequences were determined for the myxobacteria Cystobacter fuscus, Myxococcus coralloides, Sorangium cellulosum, and Nannocystis exedens and for the radioresistant bacteria Deinococcus radiodurans and Deinococcus radiophilus. A dendrogram was constructed by using weighted pairwise grouping based on these and all other previously known eubacterial 5S rRNA sequences, and this dendrogram showed differences as well as similarities compared with results derived from 16S rRNA analyses. In the dendrogram, Deinococcus 5S rRNA sequences clustered with 5S rRNA sequences of the genus Thermus, as suggested by the results of 16S rRNA analyses. However, in contrast to the 16S rRNA results, the Deinococcus-Thermus cluster divided the 5S rRNA sequences of the alpha subdivision of the class Proteobacteria from the 5S rRNA sequences of the beta and gamma subgroups of the Proteobacteria. The myxobacterial 5S rRNA sequence data failed to confirm the existence of a delta subgroup of the class Proteobacteria, which was suggested by the results of 16S rRNA analyses.

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, l-glycero-d-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained d-glycero-d-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.

A new subspecies, Staphylococcus schleiferi subsp. coagulans, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi N850274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, β-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S. schleiferi subsp. coagulans from the other coagulase-positive staphylococci is presented. The type strain is GA211 (=JCM 7470).

A new ethanol-producing bacterium, Saccharobacter fermentatus gen. nov., sp. nov., is proposed and described. This gram-negative, oxidase-negative, nonsporeforming, peritrichously flagellated bacterium ferments d-glucose to ethanol and CO2. It is H2 negative, indole negative, methyl red positive, Voges-Proskauer positive, urease negative, phenylalanine deaminase positive, and lysine decarboxylase negative. l-Arabinose, d-xylose, d-fructose, l-sorbose, d-mannitol, esculin, sucrose, maltose, and lactose are fermented with the production of acid and gas. Citrate is utilized for growth. This organism differs markedly from species in the previously described ethanol-producing genus Zymomonas. Zymomonas strains are anaerobic and degrade sugars by the Entner-Doudoroff pathway, while Saccharobacter strains are facultatively anaerobic and presumably degrade glucose by the Embden-Meyerhof-Parnas pathway. S. fermentatus was isolated from the leaves of agave in Wuhan, Hubei, People’s Republic of China. Type strain WVB8512 is preserved at the Wuhan Institute of Virology, Academia Sinica, Wuhan, Hubei, People’s Republic of China.

Strain CCHT (T = type strain), a helical procaryote that was isolated from floral surfaces of Calystegia hederacea (Convolvulaceae) in Jiangsu, People’s Republic of China, was found to be a sterol-requiring member of the class Mollicutes. The cells of strain CCHT were small, motile helices that were devoid of a cell wall or periplasmic fibrils. The organism passed through filters with average pore diameters of 300 and 220 nm. Strain CCHT grew rapidly in liquid media having simple compositions, reaching stationary growth phase within 24 h at 30°C. Growth occurred at temperatures ranging from 25 to 37°C. The organism fermented glucose and other sugars with acid production and showed weak arginine hydrolysis. It did not hydrolyze urea. In preliminary electrophoretic analyses, the cell protein patterns of strain CCHT were distinct from those of eight other spiroplasmas commonly found on flowers. Using enzyme-linked immunosorbent assays and antisera to strain CCHT, we found no serological relationship to antigens of the 23 previously recognized spiroplasma groups or species. These distinctions were confirmed by performing reciprocal metabolism inhibition and deformation serological tests, in which we used antigens and antisera to representatives of spiroplasma groups I through XXIII. The base composition (guanine-plus-cytosine content) of the DNA of strain CCHT was about 29.0 mol%. On the basis of these findings, we propose that spiroplasma strain CCH (= ATCC 43960) be designated group XXIV and the type strain of a new species, Spiroplasma chinense.

The 16S rRNA sequences of Kingella indologenes, Cardiobacterium hominis, and Bacteroides nodosus were determined by direct RNA sequencing, using a modified Sanger method. Sequence comparisons indicated that these three species represent a novel family in the gamma division of Proteobacteria. On the basis of these data, K. indologenes and B. nodosus cannot retain their current generic status as they are not closely related to other members of their assigned genera. Therefore, we propose transfer of K. indologenes to the new genus Suttonella as Suttonella indologenes and transfer of B. nodosus to the new genus Dichelobacter as Dichelobacter nodosus and assign the genera Cardiobacterium, Suttonella, and Dichelobacter to a new family, Cardiobacteriaceae, in the gamma division of Proteobacteria.

The phenotypic and chemotaxonomic characteristics of Mycoplana strains were examined. These strains were gram-negative, peritrichously flagellated, branching, filament-forming bacteria. The cellular fatty acids consisted of large amounts of straight-chain unsaturated C18:1 acid, as well as straight-chain saturated C16:0 acid and unsaturated C16:1 acid. The major ubiquinone was ubiquinone Q-10. The Mycoplana strains were divided into four groups, including one group containing Mycoplana dimorpha ATCC 4279T (= IFO 13291T = NCIB 9439T) (T = type strain) and another group containing Mycoplana bullata ATCC 4278T (= IFO 13290T = NCIB 9440T), on the basis of DNA base composition, major hydroxy fatty acid composition, mucosity of colonies, acid production from sugars, and utilization of carbon compounds. This division into groups was supported by the results of DNA-DNA homology experiments. Two new species, Mycoplana ramosa and Mycoplana segnis, are proposed. The type strain of M. ramosa is strain TK0053 (= NCIB 9440), and the type strain of M. segnis is strain TK0059 (= IFO 13240 = ATCC 21756).

Four strains of budding, hyphal bacteria, which had very similar chemotaxonomic properties, were isolated from the Baltic Sea. The results of DNA-DNA hybridization experiments, indicated that three of the new isolates were closely related, while the fourth was only moderately related to the other three. Sequence signature and higher-order structural detail analyses of the 16S rRNA of strain IFAM 1418T (T = type strain) indicated that this isolate is related to the alpha subclass of the class Proteobacteria. Although our isolates resemble members of the genera Hyphomicrobium and Hyphomonas in morphology, assignment to either of these genera was excluded on the basis of their markedly lower DNA guanine-plus-cytosine contents. We propose that these organisms should be placed in a new genus, Hirschia baltica is the type species of this genus, and the type strain of H. baltica is strain IFAM 1418 (= DSM 5838).

Phenotypic and genetic characteristics of Pseudomonas cocovenenans NCIB 9450T (T = type strain) and strains isolated from cases of food poisoning caused by consumption of fermented corn flour are compared. Our results show that these strains are members of the same species and conform to the description of Section II of the genus Pseudomonas. Because of small differences in substrate utilization patterns, the strains isolated in the People’s Republic of China are thought to be biovars of P. cocovenenans.

Four species in the order Mycoplasmatales, Mycoplasma capricolum, Mycoplasma hominis, Mycoplasma arginini, and Acholeplasma laidlawii, were compared for their ability to accumulate radiolabeled amino acids and polyamines. The use of a novel high-molecular-weight (HMW) medium, from which molecules of less than 12,000 molecular weight had been removed by extensive dialysis, allowed us to discern significant differences among the species in their relative accumulations of [3H]methionine and [3H]leucine and of [3H]spermidine and [3H]putrescine. Accumulation of radiolabeled amino acids in control low-molecular-weight (LMW) medium was small (0.2 to 2% of the label), and the species did not differ in their proportional accumulations of methionine and leucine. Accumulation of methionine was significantly enhanced (5- to 12-fold) in all species in HMW medium. In contrast, leucine accumulation was enhanced sevenfold for A. laidlawii but only twofold for M. hominis and M. capricolum in HMW medium. The nonglycolytic species, M. hominis and M. arginini, accumulated radiolabeled putrescine and spermidine in both media, whereas the glycolytic species, M. capricolum and A. laidlawii, accumulated only radiolabeled spermidine. The ability to accumulate putrescine appeared to be a differential characteristic for nonfermentative, arginine-utilizing mycoplasmas. HMW medium was much more effective than LMW medium for use in radiolabeling M. capricolum proteins with [35S]methionine.

As determined by DNA-rRNA hybridization, it is obvious that the recently described species Brochothrix campestris belongs to the genus Brochothrix. This species is closely related to Listeria species and is also related to Enterococcus faecalis, Bacillus cereus, and Bacillus subtilis. Brochothrix campestris is not closely related to the other bacteria which we tested.