- Volume 39, Issue 3, 1989

A numerical taxonomic study was performed on 52 Leuconostoc spp. strains isolated from chill-stored meats. Three clusters were observed; representative strains from these clusters, together with the type strains of previously described species, were examined by performing a cellular fatty acid analysis and deoxyribonucleic acid (DNA)-DNA hybridization. Cluster II contained seven strains, which were assigned to Leuconostoc mesenteroides subsp. mesenteroides on the basis of DNA relatedness and biochemical properties. Clusters I (30 strains) and III (15 strains) were shown to represent two new species, for which the names Leuconostoc gelidum and Leuconostoc carnosum, respectively, are proposed. The type strains of L. gelidum and L. carnosum are strains NCFB 2775 and NCFB 2776, respectively.

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. Total chromosomal deoxyribonucleic acid (DNA) for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with single-stranded unlabeled DNAs which had been immobilized on the surfaces of microdilution wells. After hybridization, biotinylated DNA was quantitatively detected with beta-D-galactosidase and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactopyranoside. Homology values obtained with this fluorometric direct binding method were compared with values obtained with two membrane filter methods, one in which photobiotin labeling was used and one in which radioisotope labeling was used. The results showed that the fluorometric direct binding method in which microdilution wells are used could be an alternative to radioisotope and membrane filter hybridization methods.

Deoxyribonucleic acid similarity experiments in which the SI nuclease methodology was used were performed on strains of Pseudomonas species, including some that had not been previously subjected to similarity measurements. The results obtained with this technique confirmed and expanded previous observations on the low levels of deoxyribonucleic acid similarity among species of the genus as determined by membrane competition experiments. A general discussion of the results and nomenclatural status of some of the species is included.

An investigation of 20 bovine and 24 ovine isolates of organisms variously described as “Histophilus ovis,” “Haemophilus agni,” and “Haemophilus somnus” revealed no consistent host-related differences in 47 biochemical characteristics; however, agarose gel electrophoresis of predominantly plasmid-derived deoxyribonucleic acid demonstrated the presence of variable numbers of plasmids of divergent size in each of the ovine strains but in none of the bovine isolates. Restriction enzyme analysis of deoxyribonucleic acids from an ovine culture and a bovine culture also suggested chromosomal differences. Our observations support the concept of one species, Histophilus ovis, comprising ovine and bovine isolates, but point to the existence of subspecific divisions related to host predilection.

A total of 22 strains of a yellow-pigmented bacterium which I designated YB, which were isolated from leaves of various plants, were compared with the type strains of Curtobacterium citreum, Curtobacterium albidum, Curtobacterium luteum, and Curtobacterium pusillum and six strains of Curtobacterium flaccumfaciens, a pathogen of soybeans and garden beans. These species comprise the genus Curtobacterium. YB was nonpathogenic when it was inoculated onto leaves of soybeans and garden beans and produced β-carotene, as did both C. citreum and C. luteum. These species and YB were all originally isolated from plants. YB could be distinguished from all previously described species of the genus Curtobacterium on the basis of more rapid production of acid from maltose and xylose, more rapid hydrolysis of gelatin, susceptibility to bacteriophage PYB-3 infection, inability to hydrolyze hippurate, growth in the presence of 10% sodium chloride, and higher deoxyribonucleic acid guanine-plus-cytosine content. Because the YB strains represent a new center of variation in the genus Curtobacterium, a new species, Curtobacterium plantarum, is proposed. This bacterium was isolated from leaves of three soybean cultivars at weekly intervals. Mean populations, expressed in colony-forming units (CFU) per gram of leaf tissue, increased gradually over the season from 28 CFU/g at 5 weeks after planting to 158 CFU/g at plant maturity. The bacterium was also isolated from leaves of 10 soybean cultivars and 10 corn inbred lines in each of 3 years, from leaves in one soybean field and one corn field in each of 25 Iowa counties, and from leaves of each of 100 soybean plants from one field and 100 corn plants from one field. Over a 2-year period, it was isolated from all 200 plant species sampled. The samples represented 62 plant families and included trees (both broad leaved, and conifers), shrubs, annuals, and perennials. The bacterium was isolated from field-grown immature and mature soybean seeds and from leaves of soybean and corn seedlings grown in a microbe-free environment from seeds treated to eliminate external microorganisms. I concluded that C. plantarum sp. nov. is seed transmitted in soybeans and corn and is ubiquitous in the leaves of plants.

The genus Prochlorothrix gen. nov., with one species, Prochlorothrix hollandica sp. nov., is proposed for filamentous oxygenic photoautotrophic procaryotes containing chlorophylls a and b. The cylindrical cells are arranged in trichomes which lack sheaths, are not motile, and do not possess differentiated cells. P. hollandica grows in liquid media or on solid mineral media and is incapable of dinitrogen fixation. Optimum growth occurs between 20 and 30°C and at pH 8.4. The deoxyribonucleic acid base composition of this organism is 53 mol% guanine plus cytosine. Emendation of the description of the order Prochlorales Florenzano, Balloni, and Materassi 1986 is proposed since the original definition of this taxon would exclude the genus Prochlorothrix. To accommodate the filamentous genera in the order Prochlorales, we propose the family Prochlorotrichaceae fam. nov.

The 16S ribosomal ribonucleic acid sequences of the type strain and three other strains of Eikenella corrodens, the type strains of Alcaligenes faecalis, Alcaligenes xylosoxydans subsp. denitrificans, Chromobacterium fluviatile, Chromobacterium violaceum, Kingella denitrificans, Kingella kingae, and Pseudomonas cepacia, and a strain of Vitreoscilla stercoraria were determined by direct sequencing of bacterial ribosomal ribonucleic acid by a modified Sanger method. These sequences were compared with previously published sequences of Neisseria gonorrhoeae and Pseudomonas testosteroni and unpublished sequences of Nitrosolobus sp., Nitrosomonas europaea, Rhodocyclus gelatinosus, Rhodocyclus purpura, Simonsiella muelleri, and Spirillum volutans. All of the bacteria sequenced in this study were members of the beta group of the class Proteobacteria, formerly called “purple bacteria and their relatives.” A phylogenetic tree was constructed based upon the sequence homologies. One of the findings of this study is that Eikenella corrodens is related, as indicated by percentage of sequence homology, to the following organisms: Kingella denitrificans (97.7%), Simonsiella muelleri (95.7%), Kingella kingae (96.2%), Neisseria gonorrhoeae (95.1%), Vitreoscilla stercoraria (94.4%), Chromobacterium violaceum (91.7%), and Chromobacterium fluviatile (89.8%). These bacteria constitute a newly recognized branch of the beta group Proteobacteria. The remaining species, including Pseudomonas cepacia, Alcaligenes faecalis, and Alcaligenes xylosoxydans subsp. denitrificans, are members of the major cluster of the beta group Proteobacteria. On the basis of our data we propose that the genera Eikenella and Simonsiella be placed in the family Neisseriaceae.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project designed to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this third report we describe numerical clusters that encompass the type strains of Mycobacterium szulgai, Mycobacterium triviale, Mycobacterium shimoidei, Mycobacterium asiaticum, Mycobacterium simiae, and Mycobacterium malmoense. Descriptions and discussion of the taxonomic status of three additional unnamed clusters, as well as a cluster that encompasses the erstwhile type strain of the presently invalid species “Mycobacterium paraffinicum,” are also presented.

Deoxyribonucleic acid-deoxyribonucleic acid hybridizations were performed with 31 vancomycin-resistant gram-positive cocci isolated from human sources. A total of 14 human isolates exhibited high levels of homology with Leuconostoc mesenteroides subsp. mesenteroides NCDO 523T (T = type strain), whereas 2 strains were genetically closely related to Leuconostoc lactis NCDO 533T. Nine human isolates formed a tight homology group with an atypical L. mesenteroides strain (strain NCIB 3351), whereas two isolates (together with four strains from food) were found to be highly related to Leuconostoc sp. strain NCDO 768T. Two new species, Leuconostoc citreum and Leuconostoc pseudomesenteroides, are proposed for these homology groups. The type strains of L. citreum and L. pseudomesenteroides are strains NCDO 1837 and NCDO 768, respectively.

Strain ELCN-1T (T = type strain), which was isolated from the hemolymph of the firefly beetle Ellychnia corrusca (Coleoptera:Lampyridae) in Maryland, was shown to be a sterol-requiring mollicute. Electron and dark-field microscopy showed that the organism consisted of small, nonhelical, nonmotile, pleomorphic coccoid cells. Individual cells were surrounded by a single cytoplasmic membrane, but no evidence of a cell wall was observed. The organism grew well in SP-4 broth medium containing fetal bovine serum, but failed to grow in formulations containing horse serum or bovine serum fraction supplements. Growth on solid media occurred only when agar cultures were incubated aerobically or in an atmosphere containing 5% carbon dioxide. Strain ELCN-1T catabolized glucose but did not hydrolyze arginine or urea. The optimum temperature for growth was 30°C, while multiplication occurred over a temperature range of 18 to 32°C. Growth was not observed at 37°C. The genome of strain ELCN-1T was determined to be about 585 megadaltons. The guanine-plus-cytosine content of the deoxyribonucleic acid was found to be 27.5 mol%. This organism was serologically unrelated to the type strains of previously described Mycoplasma species and to 18 other unclassified sterol-requiring isolates cultivated from various animal, plant, or insect sources. This is the first documented insect-derived species in the genus Mycoplasma. Strain ELCN-1 (= ATCC 43707) is the type strain of Mycoplasma ellychniae sp. nov.

Nutritionally variant streptococci (12 strains) formed two deoxyribonucleic acid hybridization groups (as determined by the SI nuclease method) that were distinct from 39 Streptococcus spp. type and reference strains. These two groups differed by their penicillin-binding protein patterns and biochemical properties. Streptococcus defectivus sp. nov. (with type strain SC10) and Streptococcus adjacens sp. nov. (with type strain GaD) are the names proposed for these species.

The taxonomic position of Bacillus subtilis strains that produce soluble black pigment is unclear. To assess the genetic relatedness between the pigmented and nonpigmented strains, deoxyribonucleic acid (DNA) reassociation was measured spectrophotometrically. Among the 40 pigmented strains examined, two distinct DNA relatedness groups were found. A total of 25 strains (group 1) showed 24 to 34% DNA relatedness and 15 strains (group 2) showed 70 to 100% relatedness to Bacillus subtilis type strain NRRL NRS-744. The intragroup DNA relatedness values for each group ranged from 85 to 100%; the intergroup relatedness values ranged from 20 to 35%. A multilocus enzyme electrophoresis analysis revealed a low level of similarity between group 1 and group 2 or the nonpigmented group. The group 2 strains and the nonpigmented strains clustered in a common group, indicating the close genetic relationship of these organisms. My results strongly suggest that group 2 is a pigmented variant of B. subtilis, but group 1 is a new species, for which the name Bacillus atrophaeus is proposed. The type strain of the new species is strain NRRL NRS-213.

The name Campylobacter pylori subsp. mustelae was recently proposed for strains belonging to the genus Campylobacter that were isolated from the gastric mucosa of ferrets because of the high levels of deoxyribonucleic acid relatedness (85 to 100%) of these strains to the type strain of Campylobacter pylori. Subsequent deoxyribonucleic acid relatedness studies done independently in three laboratories by three different methods indicate that the original results were in error. Strains isolated from ferrets in the United States, England, and Australia are members of a single species that is substantially different from C. pylori (10 to 20% relatedness at 50°C and 3 to 10% relatedness at 65°C as determined by the hydroxyapatite method; less than 10% relatedness at 65°C as determined by the nylon membrane blot method; and 30 to 49% relatedness at 66 to 68°C as determined by the spectrophotometric method). The strains isolated from ferrets also differ from human C. pylori strains in their ability to reduce nitrates, their negative leucine arylamidase reaction, their susceptibility to nalidixic acid, and their resistance to cephalothin. The strains from ferrets possess both polar and lateral flagella, whereas only polar flagella have been demonstrated in human C. pylori strains. Thus, the strains isolated from ferrets are members of a species that is distinct from C. pylon, and we propose elevating C. pylori subsp. mustelae to species status as Campylobacter mustelae sp. nov.

Selected members of the family Pasteurellaceae Pohl 1981 were investigated for their lipoquinone contents by using high-performance liquid chromatography. In addition to ubiquinones and demethylmenaquinones, menaquinones (MK-7 or MK-8 or both) were detected, mostly as minor naphthoquinone components, in several Actinobacillus, Pasteurella, and related species. Previous studies that relied on difference spectrophotometry and thin-layer chromatography did not identify menaquinone components in lipid extracts of members of the Pasteurellaceae. The view that this family can be differentiated from the Enterobacteriaceae and other fermenting gram-negative bacteria by a lack of menaquinones cannot be maintained. Although the situation seems to be more complex than previously recognized, the distribution patterns of lipoquinone structural types and their isoprenologs appear to remain a valuable chemotaxonomic tool for these bacteria.

Two new species in the genus Catellatospora are proposed. These organisms differ in carbon utilization profiles, vitamin requirements for growth, and levels of deoxyribonucleic acid relatedness to previously described species. The new species Catellatospora matsumotoense (type strain 6393-C [= IFO 14550]) contains menaquinones with 10 isoprene units (MK-10) as its major respiratory quinones, contains 3-O-methylrhamnose in its cell walls, is resistant to novobiocin, does not require thiamine for growth, and uses fructose and raffinose but not mannitol, α-methyl-D-glucoside, or rhamnose as a sole carbon source. The new species Catellatospora tsunoense (type strain 6420-P [= IFO 14552]) contains menaquines with nine isoprene units (MK-9) as its major menaquinones, does not contain 3-O-methyIrhamnose, is susceptible to novobiocin, requires thiamine for growth, and uses fructose, raffinose, and rhamnose but not mannitol or α-methyl-D-glucoside as a sole carbon source.

It has recently been proposed that three asaccharolytic species forming pigmented colonies on blood agar should be transferred from the genus Bacteroides to the new genus Porphyromonas. In the taxonomy of the genus Bacteroides cellular fatty acid profiles obtained by gas chromatography and mass spectrometry after methylation and derivatization of whole cells have proved to be useful. In this study cellular fatty acids and sugars were analyzed in strains of Porphyromonas and Bacteroides species, and the resulting multivariate data were investigated by using principal component analysis. This analysis clearly separated the Porphyromonas spp. strains from the strains of Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides intermedius, and Bacteroides levii. In the description of the genus Porphyromonas the presence of various hydroxylated fatty acids was not discussed. In this study we show that these cellular components are useful taxonomic markers.

The relationships of the yellow-pigmented hydrogen-oxidizing species Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas taeniospiralis, and “Pseudomonas carboxydoflava,” which are all members of the acidovorans ribosomal ribonucleic acid (rRNA) complex in rRNA superfamily III, were studied by using deoxyribonucleic acid (DNA):rRNA hybridization, immunotyping, numerical analysis of biochemical and auxanographic features, polyacrylamide gel electrophoresis of cellular proteins, numerical analysis of fatty acid patterns, and DNArDNA hybridization. Our results show that these five yellow-pigmented hydrogen-oxidizing Pseudomonas species are more closely related to each other than to other taxa belonging to the acidovorans rRNA complex. We propose the transfer of these species to a new genus, Hydrogenophaga, with the following four species: Hydrogenophaga flava (formerly Pseudomonas flava), Hydrogenophaga pseudoflava (to accommodate both Pseudomonas pseudoflava and “Pseudomonas carboxydoflava”), Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis), and Hydrogenophaga palleronii (formerly Pseudomonas palleronii). The type species is H. flava, with monotype strain DSM 619 (= LMG 2185 = CCUG 1658). Because H. flava grows slowly and unreliably, but is genotypically and protein electrophoretically very similar to H. pseudoflava, the latter species can be used as an alternative reference taxon for the new genus. The type strains of H. pseudoflava, H. taeniospiralis, and H. palleronii are strains GA3 (= LMG 5945 = CCUG 13799), DSM 2082 (= LMG 7170 = CCUG 15921), and Stanier 362tl (= LMG 2366tl = CCUG 20334), respectively.

Clostridium intestinalis sp. nov. is described on the basis of five strains isolated from the feces of cattle and pigs. The isolates are aerotolerant, gram-positive, sporeforming, motile rods. They differ from all the validly described aerotolerant species of the genus Clostridium by carbohydrate fermentation pattern, guanine-plus-cytosine content of the deoxyribonucleic acid, and deoxyribonucleic acid homologies. The fermentation products from peptone-yeast extract-Fildes solution-1 % (wt/vol) glucose broth are major amounts of acetic and butyric acids. The guanine-plus-cytosine content of the deoxyribonucleic acid is 26 to 28 mol%. The type strain of Clostridium intestinalis is Catt39 (= ATCC 49213 and JCM 7506).

Deoxyribonucleic acid (DNA)-DNA hybridization was performed with 10 strains belonging to the “Erwinia herbicola-Enterobacter agglomerans complex” by using the competition method on nitrocellulose filters. These strains exhibited more than 75% DNA binding to Erwinia herbicola ATCC 14589T (T = type strain) and constitute DNA hybridization group 14589 (including strains ATCC 14589T and CDC 1429-71 from DNA hybridization group III [D. J. Brenner, G. R. Fanning, J. K. Leete Knutson, A. G. Steigerwalt, and M. J. Krichevsky, Int. J. Syst. Bacteriol. 34:45–55,1984]). The high level of genomic relatedness of these strains was confirmed by the similarities observed in their electrophoretic protein patterns. On the basis of our data, DNA hybridization group 14589 constitutes a discrete species within the family Enterobacteriaceae. Its closest relative is DNA hybridization group 27155 (41 to 53% DNA relatedness), which was previously defined and includes the type strains, among others, of Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae (A. Beji, J. Mergaert, F. Gavini, D. Izard, K. Kersters, H. Leclerc, and J. De Ley, Int. J. Syst. Bacteriol. 38:77-88, 1988). We propose to unite DNA hybridization groups 14589 and 27155 in a single genus, Pantoea gen. nov. Pantoea agglomerans (Beijerinck 1888) comb. nov. is proposed to contain most strains of DNA hybridization group 27155 (including DNA hybridization group XIII of Brenner et al.), and its type strain is strain ATCC 27155 (= NCTC 9381 = LMG 1286). Pantoea dispersa sp. nov. is proposed to contain DNA hybridization group 14589, and its type strain is strain ATCC 14589 (= LMG 2603). Descriptions of the genus and its two species are given.