- Volume 41, Issue 1, 1991

A moderately halophilic actinomycete was isolated from a soil sample obtained from Death Valley, Calif. The morphological and chemical properties of the isolate conform to the characteristics of the genus Actinopolyspora, which until now has contained a single species, Actinopolyspora halophila. Our isolate can be distinguished from A. halophila on the basis of physiological and biochemical properties and DNA relatedness data. Therefore, we propose a new species for this strain, Actinopolyspora mortivallis; the type strain is strain HS-1 (= JCM 7550).

Mycoplasmas which were distinct from Mycoplasma cricetuli were isolated from the conjunctivas of Chinese hamsters. Two clones, which were derived from a single colony and were obtained on separate occasions, were examined in detail for morphology, growth, and biochemical characteristics. These clones were indistinguishable from each other and had the following properties: Guanine-plus-cytosine content of 29 mol%, requirement for sterol, facultatively anaerobic, and positive for glucose metabolism. They were serologically distinct from M. cricetuli and all 94 other previously described Mycoplasma and Acholeplasma spp. One of them, strain 128 (= NCTC 11712), is designated the type strain of a new species, Mycoplasma oxoniensis.

A pink-pigmented photoheterotrophic bacterium was isolated from lake sediment. This strain, strain 941T (T = type strain), was characterized and was found to belong to the genus Rhodopseudomonas on the basis of morphology, sessile budding mode of multiplication, and the lamellar arrangement of the internal photosynthetic membranes. Strain 941T consistently formed pink cultures, could not utilize straight-chain saturated fatty acids containing five or more carbon atoms as electron donors for photoheterotrophic growth, and was distinct from the previously described members of the genus. Strain 941 (= DSM 5909) is the type strain of a new species, Rhodopseudomonas rosea.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, “Flexispira rappini,” Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, “F. rappini,” and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and “F. rappini” were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.

A total of 120 mycoplasma strains were recovered from 97 of 265 diseased seals investigated during the seal epidemic in the North Sea and in the Baltic Sea in 1988. Mycoplasmas were isolated from the respiratory tracts (including lungs), hearts, brains, and eyes of the seals. Thirty strains were filter cloned and investigated for their morphological, biochemical, and serological characteristics compared with the characteristics of previously described species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that these strains belong to two new species, for which the names Mycoplasma phocarhinis and Mycoplasma phocacerebrale are proposed. M. phocarhinis (17 strains) did not ferment glucose or hydrolyze arginine but did reduce tetrazolium chloride and potassium tellurite and produced films and spots. M. phocacerebrale (13 strains) metabolized arginine but not glucose and produced phosphatase but did not reduce tetrazolium chloride and potassium tellurite. Both species lysed sheep erythrocytes but did not absorb sheep or guinea pig erythrocytes. The type strain of M. phocarhinis is strain 852 (= ATCC 49639), and the type strain of M. phocacerebrale is strain 1049 (= ATCC 49640).

Two mollicutes (strains F7T [T = type strain] and F28) isolated from floral surfaces of plants growing in Morocco and France were capable of sustained growth in serum-free (or cholesterol-free) mycoplasma broth media. The two isolates were found to be genomically and serologically related. Morphologic examination of the organisms by electron and dark-field microscopic techniques showed that each strain consists of small, nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of cell walls was observed. Growth in serum-free or cholesterol-free medium was sustained only when the medium contained a Tween 80 fatty acid mixture (0.01 or 0.04%). The organisms grew rapidly in most conventional mycoplasma culture medium formulations containing horse or fetal bovine serum or a bovine serum fraction and under either aerobic or anaerobic environments. The optimum temperature for growth was 28°C, but multiplication occurred over a temperature range from 20 to 35°C. Both strains catabolized glucose and mannose, but did not hydrolyze arbutin, arginine, or urea. The molecular mass of the genome of strain F7T was determined to be about 886 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was found to be 30.0 mol%. The two isolates were serologically unrelated to type strains of the 11 previously described Acholeplasma species and to 10 other unclassified sterol-nonrequiring mollicutes cultivated from various animal, plant, or insect sources. Strain F7 (= ATCC 49495) is the type strain of Acholeplasma seiffertii sp. nov.

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (<5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (>96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes. The 16S rRNA sequences of T. hyodysenteriae B78T, B204, and A-1 were nearly identical (99.8 to 99.9% base sequence similarity). T. innocens B256T and 4/71 were closely related to the T. hyodysenteriae strains (99.4 and 99.0% similarity). Strains of T. hyodysenteriae and T. innocens exhibited low levels of 16S rRNA similarity (average, 76.5%) with T. pallidum, Borrelia burgdorferi, and various other spirochetes. The results of our investigations indicate that T. hyodysenteriae and T. innocens are distinct but related species of spirochetes. T. hyodysenteriae and T. innocens are only distantly related to T. pallidum, the type species of the genus Treponema, and to other spirochetes. Consequently, we propose that the species T. hyodysenteriae and T. innocens be transferred to a new genus, Serpula, gen. nov.

Seventy-three strains of the seven recognized Listeria species were studied by performing a multilocus enzyme electrophoresis analysis of 18 enzyme loci. The mean number of alleles per locus was 9.5 and all of the loci were polymorphic. A total of 56 electrophoretic types were distinguished. Cluster analysis of a matrix of the genetic distances between paired electrophoretic types revealed that there were six principal clusters at the species level (genetic distances between clusters greater than 0.8). Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Listeria seeligeri, and Listeria ivanovii each corresponded to one of these clusters with no overlap. Our results are in agreement with those of previous DNA hybridization experiments (Rocourt et al., Curr. Microbiol. 7:383-388, 1982). Listeria grayi and Listeria murrayi electrophoretic types formed a unique cluster, thus reinforcing the suggestion of Wilkinson and Jones (J. Gen. Microbiol. 98:399-421, 1977) that these two species should be considered two biovars of a single species.

The taxonomic relationships among Rhodocyclus, Leptothrix, and Sphaerotilus species, Pseudomonas saccharophila, and Alcaligenes latus were investigated by performing DNA-rRNA hybridization experiments, by determining DNA base compositions, and by performing protein gel electrophoresis experiments. These taxa have relatively high guanine-plus-cytosine contents (67.8 to 72.5 mol%) and form a separate group within rRNA superfamily III. Rhodocyclus gelatinosus and “Leptothrix discophora” form two separate rRNA branches, which are linked at a Tm(e) level of 74.8 ± 0.7°C [Tm(e) , temperature at which 50% of an DNA-rRNA hybrid is denatured]. Also situated at this Tm(e) level and therefore equidistantly related to Rhodocyclus gelatinosus and “Leptothrix discophora” are Sphaerotilus natans, P. saccharophila, and A. latus. Leptothrix cholodnii is located on the “L. discophora” rRNA branch but produces a protein pattern which is different from that of “L. discophora.” All of the other taxa which we investigated also have unique protein patterns. Since Rhodocyclus gelatinosus is only very distantly related to the type species of the genus Rhodocyclus (Rhodocyclus purpureus) and to Rhodocyclus tenuis and because it is clearly different from its nearest phylogenetic neighbors, we propose to rename this species as Rubrivivax gelatinosus gen. nov., comb. nov. The type strain remains strain NCIB 8290 (= LMG 4311).

Sulfate-reducing bacterial strain HR100 was isolated from sediments of Retba Lake, a pink hypersaline lake in Senegal. The cells were motile, nonsporulating, and rod shaped with polar flagella and incompletely oxidized a limited range of substrates to acetate and CO2. Acetate and vitamins were required for growth and could be replaced by Biotrypcase or yeast extract. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as electron acceptors and were reduced to H2S. Growth occurred at pH values ranging from 5.5 to 8.0. The optimum temperature for growth was 37 to 40°C. NaCl and MgCl2 were required for growth; the optimum NaCl concentration was near 10%. The guanine-plus-cytosine content of the DNA was 57.1 ± 0.2 mol%. On the basis of the morphological and physiological properties of this strain, we propose that it should be classified in a new genus, Desulfohalobium, which includes a single species, Desulfohalobium retbaense. The type strain is strain DSM 5692.

A new genus and species of obligate intracellular bacterial parasite of small free-living amoebae is described. This bacterium causes fatal infections in amoebae belonging to the Acanthamoeba-Naegleria group. It does not grow on any artificial substrate deprived of living amoeba cells. The entry of the bacterium into a host occurs by phagocytosis, but growth occurs in the cytoplasm, not in phagosomes. This parasite is readily distinguished from other kinds of previously recognized bacteria that live within amoeba cells on the basis of its host cell lytic activity. The bacterium is a gram-negative, short rod with tapered ends. It multiplies intracellularly by a transverse central pinching-off process. Cells are motile by means of a polar tuft of flagella. The bacterium is surrounded by a distinct multilayered cell envelope with a chemtype A1γ peptidoglycan. The peptidoglycan is unique in its high content of glucosamine residues with free amino groups. The DNA base composition of this organism is 43 mol% guanine plus cytosine. The name Sarcobium lyticum gen. nov., sp. nov. is proposed for this bacterium. The type strain of S. lyticum is strain L2 (= PCM 2298).

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter Iari, and “Campylobacter upsaliensis.” Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus “Flexispira,” with “Flexispira rappini” as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.

Sixty rhizobial strains isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan were compared with 37 rhizobia isolated from woody legumes in other regions and with 25 representatives of recognized Rhizobium species by performing a numerical analysis of 115 phenotypic characteristics. Nineteen clusters were formed below the boundary level of 0.725 average distance, which was the level that separated the reference Rhizobium and Bradyrhizobium species. Our results indicated that tree rhizobia are very diverse with respect to their cross-nodulation patterns, as well as their physiological and biochemical properties, since 12 of the clusters formed consisted of tree rhizobia alone. Two distinctive features of tree rhizobia isolated in the Sudan were their high maximum growth temperature and their high salt tolerance.

The diversity among 191 bacterial strains isolated from stem and root nodules (151 and 40 strains, respectively) of Sesbania rostrata grown in different geographical areas in Senegal and in The Philippines was studied by using DNA-DNA hybridization techniques (S1 nuclease method), by determining DNA base compositions, by performing legume nodulation tests, and by determining nitrogenase activity. The following conclusions were drawn. (i) All of the strains produced stem and root nodules on S. rostrata. (ii) Most of the organisms (184 strains) belonged to the genus Azorhizobium; their guanine-plus-cytosine contents ranged from 66 to 68 mol%, they fixed N2 under free-living conditions, and they produced effective nodules on the stems and roots of S. rostrata. (iii) The seven other strains probably belonged to the genus Rhizobium, since guanine-plus-cytosine contents ranged from 59 to 63 mol% and they did not fix N2 under free-living conditions; three strains produced effective root nodules, but their stem nodules exhibited very low activity or were ineffective, and the four remaining strains produced ineffective nodules on both stems and roots. (iv) The genetic diversity among the 184 Azorhizobium strains allowed us to divide them into two genomic species; genomic species 1 constituted the major group (175 strains) and corresponded to Azorhizobium caulinodans since all of the strains were more than 79% related to type strain ORS 571, and genomic species 2 contained nine strains that were only 44 to 53% related to type strain ORS 571 (difference between the denaturation temperatures of homologous and heterologous hybrids, more than 6°C) and more than 76% related to reference strains SD02 and SG28 (difference between the denaturation temperatures of homologous and heterologous hybrids, less than 3°C). The species that were distinct from A. caulinodans cannot be named until they can be differentiated by phenotypic tests.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (<20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.

The name Hydrogenovibrio marinus gen. nov., sp. nov. is proposed for an obligately chemolithoautotrophic, mesophilic, gram-negative, motile, comma-shaped, aerobic, hydrogen-oxidizing bacterium that was isolated from seawater. The optimum temperature and NaCl concentration for growth are 37°C and 0.5 M, respectively. The guanine-plus-cytosine content of the DNA is 44.1 mol%. The ubiquinone is ubiquinone-8, and the major cellular fatty acids are C16:0, C18:0, and C16:1 acids. The type strain of this species is strain MH-110 (= JCM 7688).

Bacillus sp. strain DSM 2923T (T = type strain) and five other Bacillus strains isolated from soil were able to grow on nicotinate (niacin) as a sole source of carbon, nitrogen, and energy and to produce a blue pigment. The guanine-plus-cytosine contents of the DNAs of four strains ranged from 37 to 39 mol%. On the basis of the results of common physiological tests, assignment to a previously described Bacillus species was not possible. Thus, these closely related strains are described as a new species, Bacillus niacini.

An anaerobic, mesophilic, sporeforming, xylanolytic bacterium was isolated from decayed Pinus patula wood chips. The cells of this organism were gram-negative rods, had peritrichous flagella, and formed terminal round endospores which distended the cell wall. Xylan, cellobiose, fructose, glucose, maltose, mannose, melezitose, raffinose, rhamnose, salicin, sucrose, and xylose were utilized. The optimum temperature and optimum pH for growth were 35°C and 7.2, respectively. The DNA composition was 40 mol% guanine plus cytosine. The name Clostridium xylanolyticum sp. nov. is proposed for this bacterium. The type strain is strain ATCC 49623.

A new subspecies, Staphylococcus capitis subsp. ureolyticus, was isolated from human skin and is described on the basis of studies of 15 to 26 strains. DNA-DNA reassociation reactions demonstrated that these strains were closely related to Staphylococcus capitis but were significantly divergent. The strains of S. capitis subsp. ureolyticus can be distinguished from S. capitis by their positive urease activity, their ability to produce acid from maltose under aerobic conditions, their fatty acid profile, and their colony morphology. The type strain of the new subspecies is strain ATCC 49326.