- Volume 43, Issue 2, 1993

Fourteen Legionella-like strains isolated from aquatic sources have been characterized serologically, biochemically, and in terms of DNA relatedness. The strains grew on buffered charcoal-yeast extract agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for cysteine, cellular fatty acid compositions in which branched-chain acids predominate, and the possession of isoprenoid quinones of the ubiquinone series with more than 10 isoprene units in their side chains. All were nonfermentative, lacked urease, were incapable of nitrate reduction, and reacted positively with a DNA probe specific for the Legionellaceae. DNA hybridization studies in which the hydroxyapatite method was used demonstrated that the strains represented five new species of the genus Legionella. Nine of the strains were more than 90% interrelated, and the name Legionella londiniensis sp. nov. is proposed for this group. Two strains formed a second hybridization group, for which the name Legionella nautarum sp. nov. is proposed, while the three remaining species, Legionella geestiana sp. nov., Legionella quateirensis sp. nov., and Legionella worsleiensis sp. nov., are each represented by a single strain. The levels of relatedness of the new species to each other are 23% or less, and the levels of relatedness to other members of the genus ranged from 0 to 36%. L. geestiana, L. nautarum, and L. londiniensis are serologically unrelated to all other known Legionella species. L. worsleiensis cannot be separated from Legionella pneumophila serogroup 4 by serological methods and is also serologically indistinguishable from L. quateirensis; distinctions may be made on the basis of fatty acid composition and biochemical reactions.

Spiroplasmas are currently classified in a group system. Criteria for separation of the twenty-four currently designated groups include serologic relatedness, polyacrylamide gel electrophoretic patterns of proteins, guanine-plus-cytosine base ratios, and, in some cases, DNA-DNA homology. The analysis of DNA-DNA homology and serologic data from a large array of strains recently discovered in dipteran insects reveals that group VIII strain EA-1 from a syrphid fly, strain TAAS-1 from a horse fly, and group XVII strain DF-1 from a deer fly belong to a large complex (supergroup) of strains with various degrees of interrelatedness. Strains DF-1 and EA-1 share DNA-DNA homology of 33 to 48% (high-stringency conditions), while strain TAAS-1 shares 42 to 67% homology with DF-1 and EA-1. The strains had temperature optima of 30 to 37°C, but the temperature minima and maxima reflected the geographic region of strain origin. These three strains also share G+C values of about 30 mol%, utilize arginine, and tend to grow in culture to very high titers (1011 cells per ml). The helical cells of these strains are smaller than those of other spiroplasmas and readily pass through filter pores of 220 nm. These data support the taxonomic placement of the biotypes represented by strains EA-1, DF-1, and TAAS-1 into one supergroup, group VIII, with subgroups designated as VIII-1, VIII-2, and VIII-3, respectively. It is proposed that group XVII remain vacant.

Some group XVI spiroplasmas, such as strains CC-1 (Spiroplasma cantharicola) and CB-1, are associated with cantharid beetles. Fifteen related but heterogeneous strains have been isolated from mosquitoes, other insects, and a flower in France and the United States. In the present study, these seventeen strains have been compared by deformation and metabolism inhibition serological tests, by one-dimensional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by determination of the guanine-plus-cytosine content of their DNA. Five of the 17 strains were further compared by DNA-DNA hybridization and by restriction enzyme (EcoRI and HindIII) analysis of their DNA. On the basis of the resulting data, we propose that group XVI be subdivided into three subgroups. Subgroup XVI-I is represented by strain CC-1 (ATCC 43207) from a cantharid beetle in the United States, and strain MQ-6 from a wasp; subgroup XVI-2 is represented by strain CB-1 (ATCC 43208) from a cantharid beetle and two strains from mosquitoes, all in the United States; and subgroup XVI-3 is represented by strain Ar-1357 (ATCC 51126) and contains 11 strains from mosquitoes and 1 strain from a flower, all from the Savoy region of France.

The levels of genotypic relatedness among seven strains of Leuconostoc spp. isolated from Argentine raw milk, Leuconostoc lactis DSM 20202T (T = type strain) and DSM 20198, Leuconostoc mesenteroides subsp. mesenteroides NCDO 523T, Leuconostoc mesenteroides subsp. dextranicum NCDO 529T, and Leuconostoc paramesenteroides DSM 20288T were determined by performing a numerical analysis of total soluble cell protein patterns and DNA-DNA hybridization data. The Argentine raw milk strains formed a tight genotypic cluster at an r value of = 0.85 and exhibited low levels of DNA homology with the other species included in this study. These strains represent a new species within the group of leuconostocs isolated from milk or dairy products. The name Leuconostoc argentinum sp. nov. is proposed; the type strain is strain LL76.

Strains of a new type of slowly growing scotochromogenic, rose-pink-pigmented mycobacterium were isolated repeatedly from sphagnum vegetation, true moss, and soil in Ireland. These strains grew at 22, 31, and 37°C but not at 45°C and possessed acid phosphatase and arylsulfatase activities. They reduced nitrate, tolerated 0.1% NaN02, did not split amides, and were resistant to most of the antituberculous drugs tested, except ethambutol. They did not form acid from glucose and mannose. Their internal phenetic similarity was 97.08% ± 2.07%. The whole mycolate pattern confirmed the homogeneity of the taxa sharing similar mycolate types with several other mycobacterial species. However, on the basis of the nature of the major pyrolysis esters, the taxon appeared unique. The phylogenetic analysis based on evolutionary distance values revealed that the strains belong to a new species of slowly growing mycobacteria. The DNA-DNA hybridization values confirmed that these strains differ significantly from Mycobacterium nonchromogenicum, M. terrae, M. triviale, and M. thermoresistibile. The strains produced a unique rose-pink pigment and were nonpathogenic for mice, guinea pigs, and rabbits, but they provoked a nonspecific hypersensitivity reaction to bovine tuberculin in guinea pigs and cattle. Hence, they are considered a member of a new species of nonpathogenic slowly growing mycobacteria, for which the name Mycobacterium hiberniae is proposed. Strain Hi 11 is the type strain, a culture of which has been deposited in the American Type Culture Collection as strain ATCC 49874.

Five strains isolated from mussels (harvested off the Atlantic Spanish coast in 1985 and 1986) were found to be phenotypically distinct from previously described Vibrio species. They showed 94 to 100% intragroup relatedness as determined by DNA-DNA hybridization (S1 nuclease method) but were found to be only 1 to 25% related to other Vibrio species. These strains have all of the properties that define the genus Vibrio and can be clearly differentiated from other species by their positive responses in tests for Thornley's arginine dihydrolase, gas production from glucose, growth in media containing 10% NaCI, and acid production from sucrose, L-arabinose, D-xylose, and D-cellobiose and their negative responses in tests for lysine decarboxylase, the Voges-Proskauer reaction, growth without NaCI and at 40°C, hydrolysis of gelatin, casein, starch, DNA, and alginate, and acid production from D-mannose. The G+C ratio of the DNA is 45 to 46 mol%. The name Vibrio mytili is proposed for the new species; strain 165 (= CECT 632) is the type strain.

Comparative sequence analysis of in vitro-amplified 16S rRNA genes of Thiosphaera pantotropha GB17T (T = type strain) and Paracoccus denitrificans LMG 4218T revealed identical 16S rRNA primary structures for the two organisms. The level of overall DNA similarity of Thiosphaera pantotropha GB17T and P. denitrificans DSM 65T is 85%, as determined by quantitative DNA-DNA hybridization. Therefore, we propose the transfer of Thiosphaera pantotropha to P. denitrificans. The closest relative of Thiosphaera pantotropha and P. denitrificans is Thiobacillus versutus, as revealed by comparative 16S rRNA sequence analysis. These organisms are members of the alpha subclass of the Proteobacteria. Within this subclass, Thiosphaera pantotropha, P. denitrificans, and Thiobacillus versutus form a phylogenetic group with Rhodobacter sphaeroides, Rhodobacter capsulatus, and “Erythrobacter longus.”

A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

On the basis of recent phylogenetic studies of 47 species within the class Mollicutes and recent molecular and physiologic findings that distinguish a new group of non-sterol-requiring insect and plant mollicutes from Acholeplasma species, we propose a revised taxonomy of the class Mollicutes. Order I (Mycoplasmatales) is retained as described earlier (S. Razin and E. A. Freundt, p. 740–742, in N. R. Krieg and J. G. Holt, ed., Bergey’s Manual of Systematic Bacteriology, vol. 1, 1984), with provision for a single family (Mycoplasmataceae) and two genera (Mycoplasma and Ureaplasma) for classification of sterol-requiring mollicutes primarily associated with vertebrates. Emended descriptions of the order and family for the above taxa are hereby included. Proposed order II (Entomoplasmatales ord. nov.) contains subtaxa for either nonhelical (Entomoplasmataceae fam. nov.) or helical (Spiroplasmataceae) mollicutes; the latter family is hereby transferred from the order Mycoplasmatales. The new order and family permit designation and classification of a monophyletic cluster of mollicute strains associated primarily with arthropods. It is proposed that nonhelical, sterolrequiring insect and plant mollicutes previously designated Mycoplasma ellychniae (Tully et al., 1989), Mycoplasma melaleucae (Tully et al., 1990), Mycoplasma somnilux (Williamson et al., 1990), Mycoplasma luminosum (Williamson et al., 1990), and Mycoplasma lucivorax (Williamson et al., 1990) be transferred to Entomoplasma gen. nov., family Entomoplasmataceae fam. nov., as Entomoplasma ellychniae comb, nov., Entomoplasma melaleucae comb, nov., Entomoplasma somnilux comb, nov, Entomoplasma luminosum comb, nov., and Entomoplasma lucivorax comb. nov. Furthermore, the genus Mesoplasma gen. nov. within the family Entomoplasmataceae fam. nov. is proposed for four non-sterol-requiring insect and plant mollicutes. This designation requires the transfer of Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophilum (Tully et al., 1988), Acholeplasma Sciffertii (Bonnet et al., 1991), and Mycoplasma lactucae (Rose et al., 1990) to Mesoplasma florum comb, nov., Mesoplasma entomophilum comb, nov., Mesoplasma Sciffertii comb, nov., and Mesoplasma lactucae comb, nov., respectively. Previously established orders Acholeplasmatales and Anaeroplasmatales within the class Mollicutes are designated orders III and IV, respectively, in the proposed revision. Classification and characteristics of lower taxa within these two orders remain as previously given. The proposal outlined here does not consider the large cluster of nonculturable, arthropod-associated, plant-pathogenic mollicutes (“mycoplasma-like organisms”) recently shown to be more closely related to members of the order Acholeplasmatales.

Comparison of the Helicobacter nemestrinae 16S ribosomal DNA with published homologous sequences from members of the genera Helicobacter, Wolinetta, and Campylobacter reveals a close relationship between H. nemestrinae, H. pylori, and H. acinonyx. This finding is unexpected since these species differ significantly in their DNA guanine-plus-cytosine contents (24 to 38 mol%).

The taxonomy of an apparently genetically distinct gas-producing group of Bacillus strains formerly classified as Bacillus polymyxa was studied. Multilocus enzyme electrophoresis analysis confirmed the distinctiveness of the unknown taxon. Low DNA relatedness values also demonstrated that the unknown was not closely related genetically to any of the presently recognized species that have guanine-plus-cytosine values of 45 to 47 mol% or produce gas by fermentation of sugars. The unknown group was also a phenotypically distinct taxon. The data suggest that the unknown group merits recognition as a new species, for which the name Bacillus peoriae is proposed.

The name Citrobacter diversus has been rejected as a nomen dubium because it was used incorrectly by Ewing and Davis in 1972 as the name for a new species that differed in at least eight charamcteristics from the organism described by Werkman and Gillen in 1932 as Citrobacter diversum [sic].