- Volume 36, Issue 1, 1986

Deoxyribonucleic acid base composition, deoxyribonucleic acid-deoxyribonucleic acid hybridization, and biochemical studies were performed on some problematic enterococci of unknown taxonomic position. Our results indicate that some motile, nonpigmented strains are members of Enterococcus gallinarum, whereas motile, yellow-pigmented strains belong to Enterococcus casseliflavus. Four nonmotile, yellow-pigmented strains were biochemically and genetically distinct from all previously described Enterococcus species and constitute a new species, for which the name Enterococcus mundtii is proposed. The type strain of Enterococcus mundtii is strain NCDO 2375.

Bacterial strains in our culture collection similar to the recently proposed species Thermoleophilum album were examined for generic relatedness. We compared these obligately thermophilic hydrocarbonoclastic bacteria by determing the electrophoretic patterns of enzymes and soluble proteins and by using deoxyribonucleic acid-deoxyribonucleic acid hybridization techniques. Included in the hybridization study together with these strains were members of the genera Thermus, Thermomicrobium, and Bacillus. Our results confirmed that Thermoleophilum is a valid genus since no measurable reassociation was observed with the members of the other genera examined. There are two homology groups within the genus Thermoleophilum; T. album strain HS-5T (= ATCC 35263T) (T = type strain) represents the type species of the genus, and Thermoleophilum minutum sp. nov. is a second species proposed here. The type strain of T. minutum is strain YS-4 (= ATCC 35265).

The levels of cross-hybridization between deoxyribonucleic acids from Haemophilus parasuis, Haemophilus pleuropneumoniae, and unnamed Haemophilus taxon C were less than 15%. The levels of deoxyribonucleic acid complementarity between H. parasuis strains belonging to serovars 1 to 4 and polyacrylamide gel electrophoresis type I and II strains were very high (86 ± 7%), but the levels of complementarity between strain Nagasaki (serovar 5, polyacrylamide gel electrophoresis type II) and the remaining strains of H. parasuis were rather low (64 ± 5%). Our results indicate that, with the exception of serovar 5, the species H. parasuis comprises a rather homogeneous, genetically closely related group of bacteria. A further division of H. parasuis strains into subspecies according to their polyacrylamide gel electrophoresis peptide profiles is not justified on the basis of deoxyribonucleic acid sequence homologies. However, the taxonomic position of serovar 5 needs further investigation.

A new mesophilc species of bacteria, for which the name Thermomonospora formosensis is proposed, is described. This organism is characterized by a white aerial mycelium, single, heat-sensitive warty spores on both aerial and vegetative mycelia, and chemotype III/B cell walls. The type strain of Thermomonospora formosensis is strain C-36820 (= IFO 14204).

Deoxyribonucleic acid-ribosomal ribonucleic acid hybridization studies revealed that the pink-pigmented facultative methylotrophs that are in part classified as pseudomonads are not related to members of the established ribosomal ribonucleic acid homology groups of the genus Pseudomonas. The molecular data support inclusion of the pink-pigmented facultative methylotrophs in the genus Methylobacterium, as suggested by Green and Bousfield (Int. J. Syst. Bacteriol. 33:875-877, 1983).

We propose two new genera, Amycolata and Amycolatopsis, to accommodate nocardioform actinomycetes having type IV cell wall composition and lacking mycolic acids. These two genera are distinguished from each other by phospholipid and menaquinone composition and phage sensitivity. The following species were assigned to the genus Amycolata: Amycolata autotrophica comb. nov. (type strain, strain ATCC 19727), Amycolata saturnea comb. nov. (type strain, strain NRRL B16172), and Amycolata hydrocarbonoxydans comb. nov. (type strain, strain NRRL B16171). The genus Amycolatopsis includes Amycolatopsis orientalis comb. nov. (type strain, strain ATCC 19795), Amycolatopsis orientalis subsp. lurida nom. rev., comb. nov., subsp. nov. (type strain, strain NRRL 2430), Amycolatopsis mediterranei comb. nov. (type strain, strain ATCC 13685), Amycolatopsis rugosa sp. nov. (type strain, strain ATCC 43014), and Amycolatopsis sulphurea sp. nov. (type strain, strain ATCC 27624).

A total of 342 strains belonging to the genus Bacillus were examined for cytochrome oxidase activity by using a quantitative colorimetric N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase assay. The following three oxidase reaction groups were identified: (i) a highly oxidase-positive group, whose members oxidized TMPD with specific activities of more than 0.400 μmol/h per mg (dry weight); (ii) an oxidase-negative group, whose members were incapable of oxidizing TMPD (like Escherichia coli); and (iii) an intermediate group of strains designated the oxidase-indeterminate group, whose members oxidized TMPD at rates just slightly higher than the autooxidation rate. Of 293 mesophilic Bacillus strains analyzed 23% were oxidase positive, 47% were oxidase indeterminate, and 30% were oxidase negative. Of 37 thermophilic strains, 68% were highly oxidase positive. All of the psychrophiles tested (13 strains) gave oxidase-indeterminate reactions. Spectral analyses of the membranes of 58 Bacillus strains revealed that there was type c cytochrome(s) in all 39 oxidase-positive and oxidase-indeterminate strains. Cytochrome o was detected in all 58 strains, 27 strains contained cytochromes o and a+a 3, and 9 strains contained cytochromes o, a+a 3, and d. The remaining 22 strains contained cytochrome o exclusively. Analyses of 19 oxidase-negative strains revealed that 17 lacked type c cytochromes; the exceptions were Bacillus megaterium ATCC 14581T (T = type strain) and Bacillus pantothenticus No. 1T. All 19 oxidase-negative strains contained cytochrome o; 11 of these strains also contained cytochromes a+a 3, 4 strains contained cytochromes o, a+a 3, and d, and another 4 strains possessed cytochrome o exclusively as the sole terminal oxidase.

A new actinomycete genus, Kibdelosporangium, is described. Members of this genus have type IV cell walls (meso-diaminopimelic acid, d-glutamic acid, dl-alanine, muramic acid, N-acetyl-D-glucosamine, d-galactose, and arabinose) and a type A whole-cell sugar pattern (d-galactose and arabinose) plus traces of madurose (3-O-methyl-D-galactose). No mycolic acids are present. The substrate mycelium has a tendency to fragment. The aerial mycelium bears long chains of spores, as well as sporangiumlike structures with well-defined walls. These sporangiumlike structures do not contain spores but germinate directly when they are placed on agar. The type strain of K. aridum is strain SK&F-AAD-216 (= ATCC 39323).

Seven arginine-utilizing mycoplasmas isolated from upper respiratory tracts of normal swine were shown to be serologically distinct from other arginine-utilizing species previously isolated from swine. The isolates required sterol for growth but did not utilize glucose or urea. The isolates were distinct from over 80 additional mycoplasmas, as determined by epiimmunofluorescence and growth inhibition tests. Based on morphological, biological, and serological properties, we propose that these strains constitute a new mycoplasma species, Mycoplasma hyopharyngis. Strain H3-6B F (ATCC 35707) has been designated as the type strain.

A total of 22 species of the order Mycoplasmatales, representing eight serologically distinct groups, were tested for the presence of the first two enzymes of the arginine dihydrolase pathway by using two-dimensional thin-layer chromatography to separate end products. Both citrulline, produced by arginine deiminase, and ornithine, produced by ornithine carbamoyltransferase, were found in the glycolytic serogroup 1 species Mycoplasma putrefaciens, Mycoplasma capricolum, and Mycoplasma sp. bovine group 7, in Mycoplasma fermentans (serogroup 6), and in the nonfermentative serogroup 7 species Mycoplasma arginini, Mycoplasma hominis, Mycoplasma gallinarum, Mycoplasma gateae, Mycoplasma salivarium, Mycoplasma orale, and Mycoplasma buccale. Citrulline alone was found in one glycolytic species, Mycoplasma gallisepticum (serogroup 4). No end products were found in six other fermentative Mycoplasma species, in three Acholeplasma species, or in two Ureaplasma urealyticum serovars. The use of radiolabeled substrate with representative species showed that citrulline and ornithine were produced from arginine and not derived from the medium.

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate (i.e., 0.5%), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-salts medium containing 0.005% yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production. A type culture has been deposited with the American Type Culture Collection, Rockville, Md. (ATCC 35960).

We determined the levels of ribosomal ribonucleic acid similarity among 30 Bacteroides species. The ribosomal ribonucleic acid homology values (determined by using the membrane competition method and both 16S and 23S ribosomal ribonucleic acid subunits) ranged from 100 to 4%. The organisms presently placed in the genus Bacteroides represent a major phylogenetic unit, as shown by the sequence diversity of the ribosomal ribonucleic acid cistrons among the species. There were three major clusters of species, which had intercluster homology values of 20 to 30%. The first cluster consisted of Bacteroides fragilis and nine other saccharolytic species which are common fecal organisms. The second cluster contained Bacteroides melanogenicus and 11 other moderately saccharolytic species, many of which are commonly isolated from human oral cavities. The third, rather heterogeneous cluster contained Bacteroides asaccharolyticus and four other mostly asaccharolytic species.

Ribosomal ribonucleic acid (RNA) homology studies indicated that there is 90 to 96% homology between Azospirillum lipoferum and Azospirillum brasilense and 64 to 70% homology between these two species and Azospirillum amazonense. These findings support the inclusion of these three species in the genus Azospirillum. In contrast, “Azospirillum seropedicae” strains showed very little homology with the other Azospirillum species (< 22% RNA homology) and should not be considered a member of this genus. The taxonomic placement of “Azospirillum seropedicae” is uncertain. The nearest relatives of the genus Azospirillum were Aquaspirillum itersonii and Rhodospirillum rubrum (>65% RNA homology); Gluconobacter oxydans and Beijerinckia indica exhibited 30 to 60% RNA homology with Azospirillum species. Deoxyribonucleic acid studies indicated that Conglomeromonas largomobilis subsp. largomobilis was related to Azospirillum lipoferum at a level of deoxyribonucleic acid homology of >45% and at a level of RNA homology of 99%; moreover, this organism was found to be a microaerophilic nitrogen fixer. Thus, C. largomobilis subsp. largomobilis is a subjective synonym of Azospirillum lipoferum. In contrast, deoxyribonucleic acid homology studies indicated that C. largomobilis subsp. parooensis is not related to C. largomobilis, Azospirillum lipoferum, or any other species tested, and its taxonomic position is uncertain. Several strains of azospirilla which form unique star-shaped colonies were identified as Azospirillum lipoferum by deoxyribonucleic acid homology.

During a survey of the occurrence of Azospirillum spp. in cereal roots, we obtained 119 isolates which could not be identified as members of one of the three previously described Azospirillum species. These strains formed a very homogeneous group of N2-fixing, microaerobic, motile, vibrioid, gram-negative rod-shaped organisms which formed a veillike pellicle in semisolid medium similar to that of Azospirillum spp. However, the new isolates differed from Azospirillum spp. by their smaller cell width (0.6 to 0.7 μm), variable flagellation (one to three flagella on one or both poles), moist brownish colonies, and broader pH and oxygen tolerance for nitrogenase activity. Organic acids were the preferred carbon sources, but glucose, galactose, l-arabinose, mannitol, sorbitol, and glycerol were also used. The guanine-plus-cytosine content of the deoxyribonucleic acid was slightly lower than the guanine-plus-cytosine contents of Azospirillum spp. (66 to 67 mol%). Deoxyribonucleic acid hybridization experiments with 17 strains of the group showed 50 to 100% complementarity, while the levels of hybridization with the type strains of Azospirillum brasilense, Azospirillum lipoferum, and Azospirillum amazonense were 23, 15, and 6%, respectively. For these new isolates we propose a new genus, Herbaspirillum (the name refers to the habitat of the organisms, the roots of cereals, which are herbaceous seed-bearing plants). The type species is named Herbaspirillum seropedicae after the place where it was first isolated. The type strain is strain Z67, which has been deposited in the American Type Culture Collection as strain ATCC 35892.

Genetic transformation studies were used to determine relatedness within the family Pasteurellaceae. Among strains with < 60% relatedness to Haemophilus influenzae based on deoxyribonucleic acid hybridization, two groups were identified; one, showing competition for homospecific transformation with H. influenzae, contained Haemophilus parainfluenzae, Haemophilus parasuis, Haemophilus aphrophilus, Haemophilus paraphrophilus, Pasteurella pneumotropica, Pasteurella multocida, and Actinobacillus actinomycetemcomitans, and the other, showing little or no competition for homospecific transformation with H. influenzae, contained Haemophilus ducreyi, Haemophilus parahaemolyticus, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus pleuropneumoniae, and Pasteurella ureae. Such groupings support existing studies which have used only deoxyribonucleic acid hybridization or numerical analysis.

We propose that Peptococcus heliotrinreducens Lanigan 1983 be transferred to the genus Peptostreptococcus as Peptostreptococcus heliotrinreducens. An emended description is provided. The type strain is strain RHS1 (= NCTC 11029 = ATCC 29202).

I propose that “Nocardia aerocolonigenes” be placed in the genus Saccharothrix as Saccharothrix aerocolonigenes because it exhibits the following chemotaxonomic properties characteristic of this genus: Whole-cell hydrolysates contain the meso isomer of diaminopimelic acid, rhamnose and galactose are the characteristic sugars, and mycotic acids are absent. This species is phenotypically different from the type species of Saccharothrix, Saccharothrix australiensis (strain NRRL 11239), and deoxyribonucleic acid from the type strain of S. aerocolonigenes was determined to be only 10% homologous to deoxyribonucleic acid isolated from S. australiensis NRRL 11239. A description of the species is presented. The type strain is strain NRRL B-3298 (= ISP 5034 = ATCC 23870 = IFO 3837 = Shinobu No. 701).

Enterococcus hirae and “Streptococcus durans” homology group II were found to be highly related by using deoxyribonucleic acid-deoxyribonucleic acid hybridization and well-characterized strains. The “S. durans” homology group II strains used in this study were from cattle feces and human sputum. Therefore, we conclude the “S. durans” (homology group II) belongs to the same taxon as E. hirae and that the habitat of this species includes human and cattle sources.