- Volume 36, Issue 2, 1986

14C-labeled ribosomal ribonucleic acids (rRNAs) were prepared from Bradyrhizobium japonicum NZP 5549T(T = type strain) and Rhodopseudomonas palustris DSM 130, and [3H]rRNAs were prepared from Rhizobium meliloti NZP 4009 and Rhizobium loti NZP 2037. Labeled rRNA from Agrobacterium tumefaciens ICPB TT111 (a member of Agrobacterium cluster 2) was also used. These rRNAs were hybridized under optimal conditions with filter-fixed deoxyribonucleic acids (DNAs) from 24 strains of rhizobia representing nine previously identified DNA-DNA homology groups and with DNAs from various other organisms. Each hybrid was described by the following two parameters: the temperature at which 50% of the hybrid was denatured (Tm(e) ) and percentage of rRNA binding. From rRNA similarity maps and a Tm(e) dendrogram the following conclusions were drawn. (i) There are at least three genetically distinct groups in the present genus Rhizobium. Group I includes Rhizobium meliloti, Rhizobium fredii, and Rhizobium leguminosarum. Group II is represented by Rhizobium loti, and group III includes rhizobia isolated from Galega officinalis and Galega orientalis (goatsrue.). (ii) The rRNA cistrons of group I strains resemble those of Agrobacterium cluster 2 strains. (iii) The rRNA cistrons of Rhizobium fredii resemble those of Rhizobium meliloti more closely than the rRNA cistrons of Rhizobium leguminosarum or Agrobacterium cluster 2 do. (iv) The rRNA cistrons of Rhizobium loti resemble the rRNA cistrons of Rhizobium meliloti less than the rRNA cistrons of strains belonging to either Agrobacterium cluster 1 or Agrobacterium cluster 2. (v) The rRNA cistrons of Galega rhizobia resemble the rRNA cistrons of Rhizobium meliloti about as much as the rRNA cistrons of Agrobacterium cluster 1 and resemble the rRNA cistrons of Rhizobium loti less. (vi) The rRNA cistrons of Rhizobium and Bradyrhizobium resemble one another much less than do the rRNA cistrons of Rhizobium and other genera presently included in the family Rhizobiaceae (Agrobacterium and Phyllobacterium). (vii) Bradyrhizobium strains belonging to four DNA-DNA homology groups all contain rRNA cistrons which closely resembled the rRNA cistrons of Bradyrhizobium japonicum NZP 5549T. (viii) B. japonicum contains rRNA cistrons which have diverged from the rRNA cistrons of Rhodopseudomonas palustris to about the same extent as the rRNA cistrons of Rhizobium group I and Agrobacterium; on this basis we suggest that the photosynthetic ancestor of Bradyrhizobium may have been a rhodopseudomonad and that a phylogenetic classification should group these genera together. (ix) Stemnodulating strain ORS 571, from Sesbania rostrata belongs to the Rhodopseudomonas palustris rRNA branch; it is quite distinct from B. japonicum and Rhodopseudomonas palustris. Two representative strains which nodulate roots belong in the group containing the fast-growing rhizobia.

Acidiphilium organovorum sp. nov. is a gram-negative, heterotrophic bacterium that was isolated from a culture of Thiobacillus ferrooxidans which had been grown autotrophically on FeSO4-basal salts medium for several years. Purification of A. organovorum was carried out on a 1.0% glucose-basal salts medium (pH 3.0) solidified with agarose. Growth was enhanced by adding high concentrations of glucose (0.5 to 2.0%) and by supplementing the medium with yeast extract and trace amounts of FeSO4. However, these supplements were not necessary for growth. A wide variety of organic compounds were suitable substrates for growth, but inorganic forms of reduced sulfur or ferrous iron were not. Doubling times of 2.5 h and cell densities of >2 × 1010cells per ml were obtained at the optimal temperature of 37°C and pH 3.0. The guanine-plus-cytosine content of the deoxyribonucleic acid was 64 mol%. A. organovorum contains at least three distinct plasmids; one of these plasmids is larger than 30 kilobase pairs, and two are smaller than 4.0 kilobase pairs. Homology studies in which we compared the total deoxyribonucleic acid of A. organovorum with the total deoxyribonucleic acids of Acidiphilium cryptum and several Thiobacillus species indicated that A. organovorum is most closely related to A. cryptum. A. organovorum can be differentiated from Thiobacillus acidophilus by its higher temperature optimum, its faster growth rate, and its inability to utilize reduced forms of sulfur or iron as energy sources. The abundant cell growth that occurs in a medium which either is rich in organic compounds or completely lacks nutritional supplements distinguishes A. organovorum from A. cryptum. The other physiological and genetic characteristics which we examined are in close agreement with the characteristics of members of the genus Acidiphilium. The type strain of A. organovorum is strain ATCC 43141.

Eighteen strains of anaerobic gram-positive rods exhibiting an unusual three-dimensional cellular arrangement resembling test tube brushes (TTB) were recovered from subgingival dental plaque samples of randomly selected periodontitis patients. The isolates were obtained from subgingival sites exhibiting bone loss of ≥20% and pocket depth of ≥4 mm. Based upon biochemical profiles, these organisms belong to the family Propionibacteriaceae and are somewhat similar biochemically to some species of Eubacterium, notably Eubacterium saburreum. Relatedness between TTB bacteria and E. saburreum was assessed by deoxyribonucleic acid base composition and deoxyribonucleic acid renaturation rates. Moles percent guanine plus cytosine were 32 and 33, respectively. Hybridization studies showed 32% relatedness between E. saburreum and TTB bacteria, indicating that they are related but distinct species. We therefore propose a new species, Eubacterium yurii. Strains of this species characteristically form three-dimensional brushlike aggregates mediated by an amorphous, extracellular material. E. yurii is divided into two subspecies that exhibit 53% homology: E. yurii subsp. yurii, which is phosphatase positive, and E. yurii subsp. margaretiae, which is phosphatase negative.

3H-labeled rRNA was prepared from Acinetobacter calcoaceticus ATCC 23055Tand hybridized with filter-fixed DNA from carefully selected Acinetobacter strains, reference strains of taxonomically well-located genera, or some selected misnamed strains. The hybridization parameters, the temperature at which 50% of the hybrid was denatured (Tm(e)) and percent rRNA binding, were determined. The following conclusions can be drawn from the DNA:rRNA hybridization results, the rRNA similarity map, and a Tm(e) dendrogram. (i) The genus Acinetobacter is rather heterogeneous. It covers a 5°C Tm(e) range. (ii) A combination of our hybridization data and data from the literature indicates that several additional strains belong in the genus Acinetobacter. (iii) Acinetobacter Iwoffii ATCC 17960 is certainly not a member of the genus Acinetobacter. (iv) The genus Acinetobacter is not related to one of the well-established genera or families, nor is it a true member of one of the previously described rRNA superfamilies. The genus Acinetobacter forms a new and separate rRNA branch which is taxonomically slightly more close to rRNA superfamily II (Pseudomonas fluorescens complex, Azomonas, Azotobacter, etc.) than to rRNA superfamily I (Enterobacteriaceae, Vibrionaceae, Pasteurellaceae, etc.) and the genus Xanthomonas. (v) The misnamed species “Pseudomonas pavonacea” and “Pseudomonas cruciviae” belong to the Acinetobacter rRNA branch, although they differ from the genus Acinetobacter in several important phenotypic features.

We propose the new genus and species Chryseomonas polytricha for a group formerly called Ve-1. Each of the nine strains in the new species was examined for 129 characteristics, including reactions in 58 enzyme tests (API ZYM System; API Systems SA). These bacteria are rod shaped, aerobic, gram negative, yellow pigmented, motile by multitrichous polar flagella, and oxidatively saccharolytic, but they do not produce cytochrome oxidase. The mean guanine-plus-cytosine content of the deoxyribonucleic acid (DNA) of three representative strains is 54.6 ± 1.1 mol%. The DNA relatedness of eight strains of C. polytricha to the type strain E2770 (= NCTC 11843) averaged 95% (hydroxyapatite method, 60°C). Relatedness to the type strains of the five major ribosomal ribonucleic acid hybridization groups of Pseudomonas and to a reference strain of group Ve-2 was 2% or less. Most of the strains of the new species were isolated from human clinical specimens.

A new species of the genus Dactylosporangium is described, for which the name Dactylosporangium fulvum is proposed. In addition to finger-shaped sporangia, this organism produced abundant globose bodies and coremia. It differs significantly from previously described species of the genus Dactylosporangium in its cultural and physiological characteristics. The type strain of D. fulvum is SF-2113 (IFO 14381).

Nine strains of spiroplasma subgroup 1-3, which comprise the etiological agent of corn stunt disease, were similar in their serological properties. Strain E275T(T = type strain) was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Mollicutes for descriptions of new mollicute species. This strain was shown to belong to the class Mollicutes by the ultrastructure of its limiting membrane, its procaryotic organization, its colonial morphology, and its filtration behavior and to the family Spiroplasmataceae by its helical morphology and motility. Although some serological cross-reactions with other group I spiroplasma strains was observed, strain E275Tcould be readily distinguished from representatives of other group I subgroups. Subgroup 1-3 spiroplasmas and other group I strains also differed in their one- and two-dimensional polyacrylamide gel protein patterns, plant and insect host ranges, and pathogenicities. Growth in MIA or MID medium occurred at 20 to 30°C. Cholesterol was required for growth. Glucose was fermented, and arginine was catabolized. Subgroup 1-3 strains, including strain E275T, reacted with considerable homogeneity in deformation tests and were completely separable from strains belonging to subgroup I-1 (Spiroplasma citri) and subgroup I-2 (Spiroplasma melliferum). Strain E275Twas also serologically distinct from subgroups I-4 through I-8, Spiroplasma floricola (group III), Spiroplasma apis (group IV), Spiroplasma mirum (group V), and representative strains of spiroplasma groups II and VI through XI. The deoxyribonucleic acid of strain E275Thybridized with the deoxyribonucleic acid of S. citri at significant levels (33 to 68%, depending on the technique used). These results demonstrate that strain E275Tand similar strains meet the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee for elevation of spiroplasma subgroups to species. We propose that such strains be named Spiroplasma kunkelii. Strain E275Thas been deposited in the American Type Culture Collection as strain ATCC 29320T.

A new species of Actinomadura, which was isolated from soil collected in India, is characterized by sparse aerial mycelia, yellowish gray to brown vegetative mycelia, a carbohydrate utilization pattern of adonitol, cellobiose, glucose, and ribose, unique physiological characteristics, and the production of a new polyether antibiotic. Whole cells contain meso-diaminopimelic acid and madurose, but do not contain nitrogenous phospholipids or mycolic acids. The name proposed for this new species is Actinomadura oligospora, and the type strain is strain A80190.1 (= NRRL 15878).

Eubacterium desmolans sp. nov., an obligate anaerobe with desmolytic activity, was isolated from cat feces. Desmolase, which was previously observed only in a Clostridium species isolated from human fecal flora, cleaves the carbon-carbon bond of 17-hydroxylated corticoids between C-17 and C-20, thereby converting these compounds to androstenes (C19 steroids). In primary cultures on sheep blood agar plates, E. desmolans forms nonhemolytic, circular to irregular, convex, shiny, entire, white to colorless colonies that are barely visible to the unaided eye. The cells are plump, short rods that are 0.8 to 1.1 μm wide by 1.7 to 2.3 μm long and have capsules and flagella. They occur singly or in short chains and are gram positive. Inositol is the sole carbohydrate fermented. Indole is produced. The major fermentation products in inositol-enriched media are acetic and butyric acids. The type strain is strain ATCC 43058.

A new, antibiotic-producing species of Actinoplanes, Actinoplanes caeruleus, was isolated from soil. This organism formed irregular to globose sporangia which upon wetting dehisced to release spherical to oval, polarly flagellated motile spores. It contained arabinose and xylose as characteristic whole-cell sugars. This organism differed from other species of the genus Actinoplanes by forming deep blue vegetative mycelial pigments, by the absence of diaminopimelic acid on the cell wall, by its ability to hydrolyze adenine and hypoxanthine, by its resistance to lysozyme, and by its inability to utilize l-arabinose, d-xylose, and succinate as sole carbon sources. The type strain of A. caeruleus is strain SCC 1014 (= ATCC 33937).

The membrane protein profiles of marine budding bacteria belonging to the genus Hyphomonas were determined and analyzed. Hyphomonas spp. synthesized an unusually large number (four or more) of high-apparent-molecular-weight proteins (apparent molecular weight, ≥64,000). Species were compared by using a membrane protein profile index developed for this study. Our results supported the division of extant members of this genus into five species. The membrane protein profile index for Hyphomonas hirschiana and Hyphomonas neptunium was higher than suggested by other phenetic characteristics.

Acidophilic heterotrophic bacteria recovered from samples of water and sediment collected from acidic mine drainage streams were compared nutritionally, genetically, and morphologically. All 37 bacterial strains examined were rod shaped, motile, gram negative, and strictly aerobic, utilized citric acid and Tween 80 as sole carbon sources, and were unable to grow at or above pH 6.0. The ultrastructure of representative strains was not markedly different from that of gram-negative bacteria. Differences among the strains were evident in cell size (4.2 by 0.6 to 1.2 by 0.6 p.m), pigmentation (when present), and nutritional faculties (the carbon sources suitable for growth of individual strains ranged from 8 to 20 of the 32 compounds tested). The guanine-plus-cytosine base composition of eight typical strains ranged from 63 to 68 mol%. All of the strains exhibited primary characteristics of the recently described genus Acidiphilium; however, important differences between our strains and the type species Acidiphilium cryptum suggested that new Acidiphilium species should be described. No significant deoxyribonucleic acid-deoxyribonucleic acid homology was found between five acidophilic heterotrophic strains and A. cryptum. Furthermore, no significant deoxyribonucleic acid-deoxyribonucleic acid homology was evident between the acidophilic heterotrophs and six Thiobacillus species. The bacteria which we studied could be divided into three groups based on genetic and nutritional characteristics. We propose the following three new species: Acidiphilium rubrum (type strain, strain OP [= ATCC 35905]), Acidiphilium angustum (type strain, strain KLB [= ATCC 35903]), and Acidiphilium facilis (type strain, strain PW2 [= ATCC 35904]).

The radiation-resistant, red-pigmented bacteria belonging to the genus Deinococcus appear phenotypically similar to Micrococcus roseus, but the complex envelope profile and biochemical phylogenetic markers show them to be gram-negative clones of ancient lineage. All the type strains of indubitable Deinococcus species (D. radiodurans, D. radiophilus, D. proteolyticus and D. radiopugnans) and all the representative strains of D. radiodurans have unusual numbers of polar lipids (mostly phosphoglycolipids) and do not have phosphatidylglycerol (PG) or the phospholipids derived from it. This polar lipid profile is distinctive of the genus, and the patterns are useful for characterization. The lack of PG or di-PG and their derivatives provides a reasonably easy assignment to the genus Deinococcus and may allow recognition of relatives within the Deinococcaceae, which may not be radiation resistant. The species incertae sedis, D. erythromyxa, has a normal form of phospholipid profile, including PG. This, together with the peptidoglycan type L-Lys-L-Ala3-4, forces exclusion from Deinococcus and reassignment to Micrococcus.

The catalase activities of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains were measured. M. intracellulare and M. avium had significantly lower activities than M. scrofulaceum. The percentage of catalase activity remaining after exposure of cell-free extracts from late-log-phase cells to 53°C for 50 min allowed differentiation among the three species; M. intracellulare catalase retained 14.1 ± 7.9% (mean ± standard deviation) of its activity, M. avium retained 53.3 ± 7.4% of its activity, and M. scrofulaceum catalase was very resistant and retained 82.8 ± 6.7% of its activity. Cells of all three species harvested in stationary phase exhibited higher percentages of heat-resistant catalase, and species could not be differentiated at this stage in the growth cycle. Polyacrylamide gel electrophoresis of crude extracts from late-log-phase cells produced two bands of catalase activity in both M. intracellulare and M. avium extracts and four bands of activity in M. scrofulaceum extracts stained by diaminobenzidine. These bands differed in their susceptibilities to heat inactivation and inhibition by 3-amino-1,2,4-triazole.

The characteristics of a group of slow-growing, fusiform, fastidious anaerobes isolated from advanced periodontal lesions in human oral cavities were examined. Our results indicated that 12 fusiform Bacteroides strains belong to a new species in the genus Bacteroides. The name Bacteroides forsythus is proposed for these isolates. The type strain is strain ATCC 43037.

A new species belonging to the photosynthetic bacterial genus Chromatium is described. This new organism differs from all other Chromatium species in its thermophilic character and hot-spring habitat. In addition, the combination of its carotenoid pigments, physiological peculiarities, and deoxyribonucleic acid base composition clearly define this isolate as a new species of photosynthetic purple bacteria. The organism is a rod-shaped, gram-negative bacterium which produces bacteriochlorophyll ap and grows photoautotrophically with sulfide as an electron donor at an optimum temperature of 48 to 50°C. No growth is observed below 34°C or above 57°C. Globules of elemental sulfur are produced from the oxidation of sulfide and are stored intracellularly. Acetate and pyruvate are the only organic compounds that are photoassimilated. The major carotenoids of the new organism are rhodovibrin and spirilloxanthin, and the deoxyribonucleic acid base composition is 61 mol% guanine plus cytosine. Based on these characteristics, I propose a new species, Chromatium tepidum; the specific epithet refers to the moderately thermophilic nature of this hot-spring photosynthetic bacterium.

Using deoxyribonucleic acid hybridization (S1 nuclease method), we identified 12 hybridization groups (genospecies) containing 74 strains among 85 Acinetobacter strains studied. A total of 28 characters which allowed identification of the genospecies were applied to 266 strains. Of the 12 genospecies, 11 could be unambiguously identified. Genospecies 1 (Acinetobacter calcoaceticus sensu stricto) contained eight glucose-oxidizing strains which were unable to grow at 44°C and were isolated from soil, including the type strain of A. calcoaceticus. Genospecies 2, which contained 121 strains (mostly glucose oxidizers that were able to grow at 44°C), was named Acinetobacter baumannii sp. nov. (type strain, strain ATCC 19606). Genospecies 3 contained 15 strains that were able to oxidize glucose and to grow at 41°C but not at 44°C. Genospecies 4, which contained 23 hemolytic and proteolytic strains that were able to utilize dl-4-aminobutyrate but not dl-lactate, was named Acinetobacter haemolyticus sp. nov. (type strain, strain ATCC 17906). Genospecies 5, which contained 17 strains that were unable to oxidize glucose and able to utilize dl-lactate and l-histidine but not glutarate or azelate, was named Acinetobacter junii sp. nov. (type strain, strain ATCC 17908). Genospecies 6 contained only three hemolytic, proteolytic strains that were unable to utilize dl-lactate, malonate, or dl-4-aminobutyrate. Genospecies 7, which contained 23 strains that were unable to grow at 37°C and to oxidize glucose and utilized only a few carbon sources was named Acinetobacter johnsonii sp. nov. (type strain, strain ATCC 17909). A total of 34 strains had the characteristics of genospecies 8/9 (mostly glucose negative; utilized azelate but not Simmons citrate, glutarate, l-histidine, l-aspartate, l-leucine, β-alanine, or 2,3-butanediol). Genospecies 8 was Acinetobacter lwoffii sensu stricto since it contained the type strain of this species. Genospecies 9 could not be differentiated from genospecies 8. Genospecies 10 (four strains), 11 (four strains), and 12 (three strains) were differentiated by their nutritional patterns.

The levels of genetic relatedness of 19 Hyphomicrobium strains which utilize one-carbon compounds were determined by deoxyribonucleic acid (DNA)-DNA hybridization in solution under optimal conditions (S1 nuclease technique). Most of these hyphomicrobia fell into four groups with high levels of relatedness (level of homology within each group, 86 to 110%). These groups were only distantly related to each other (levels of homology between groups, 1 to 9%). Three additional groups of C1-utilizing hyphomicrobia were represented by only one strain each. In addition, the levels of DNA-DNA homology of four Hyphomonas species and 11 Hyphomicrobium-Hyphomonas-like isolates were determined. Seven of these isolates formed three groups containing two or three strains each; the level of homology within each group was 94 to 120%. These groups of peptide-utilizing strains were related to Hyphomonas spp. at a DNA-DNA homology level of 13 to 43%; thus, they represented new species of Hyphomonas. Four of the isolates had less than 10% DNA homology with either Hyphomicrobium reference strains or Hyphomonas spp. Strain B-1408 was distinguished by its DNA base composition of 46.19 mol% guanine plus cytosine, which is 14 mol% below the average base composition of Hyphomicrobium spp. or Hyphomonas spp. The levels of genetic relatedness of other hyphal, budding bacteria, such as Pedomicrobium spp., genus F, Rhodomicrobium vannielii, and genus T, to Hyphomicrobium sp. strain MC-750 were too low to be evaluated by DNA-DNA hybridization techniques.