- Volume 40, Issue 2, 1990

Two strains of Methanogenium bourgense, strains MS2T (T = type strain) and LX1, were characterized, and, based in part on previously published DNA hybridization data, this species was transferred to a new genus, Methanoculleus, as Methanoculleus bourgense comb. nov. Methanogenium marisnigri JR1T and a new strain of Methanogenium marisnigri, strain AN8, were also characterized. This species was also transferred to the genus Methanoculleus as Methanoculleus marisnigri comb. nov. et emend., and its description was emended to indicate that the species has a temperature optimum near 40°C, is halotolerant, and is slightly alkaliphilic; in contrast, the previous description of this organism indicates that it has a temperature optimum of 20 to 25°C, is halophilic, and is slightly acidophilic. We also propose the transfer of two other phylogenetically related species, Methanogenium thermophilicum and Methanogenium olentangyi, to the genus Methanoculleus as Methanoculleus thermophilicum and Methanoculleus olentangyi, respectively. Methanogenium cariaci JR1T was also further characterized, and its description is emended.

A new species, Bacillus naganoensis, is proposed for an obligately aerobic, moderately acidophilic, endospore-forming bacterium that produces a thermostable, aciduric pullulanase (EC 3.2.1.41). The organism was isolated from soil by selection on solid, pullulan-containing medium at pH 4.0 and 30°C. The isolate required a medium pH of less than 6.5 for growth initiation. Fatty acid composition studies revealed that the major fatty acid of cells grown in nutrient broth supplemented with 1% starch was 14-methylpentadecanoic acid (iso-C16) at 45 mol%. The guanine-plus-cytosine content of the DNA of this organism was 45 ± 2 mol%. A type culture has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 53909.

We performed hybridizations between labeled rRNAs from seven representative members of the family Pasteurellaceae and from three other taxa on the one hand and DNAs from 53 strains known or presumed to belong to the Pasteurellaceae on the other hand. The members of the Pasteurellaceae are most closely related to members of the Enterobacteriaceae, the Vibrionaceae, the Aeromonadaceae, and the genus Alteromonas. The family Pasteurellaceae is very heterogeneous. There are at least seven rRNA branches. Several organisms with the same genus name are dispersed over the entire dendrogram. The “Histophilus ovis,” [Haemophilus] ducreyi, [Actinobacillus] actinomycetemcomitans, and [Haemophilus] aphrophilus rRNA branches are separate and quite remote from the three authentic genera in this family; this might justify eventual later separate generic status. DNA-rRNA hybridization with suitable, labeled rRNA probes is an excellent method to establish whether an organism belongs in the Pasteurellaceae; e.g., some strains of Bisgaard’s taxa 7, 13, and 16 and of the gas-producing “SP” group certainly belong in this family, whereas three bovine lymphangitis organisms (strains NCTC 10547, NCTC 10549, and NCTC 10553), [Haemophilus] piscium ATCC 10801T (T = type strain), and [Pasteurella] piscicida ATCC 17911 belong in the Enterobacteriaceae, the Aeromonadaceae, and the Vibrionaceae, respectively.

Strain 831-C4T (T = type strain), isolated from the surface of lettuce plants (Lactuca sativa) obtained from a retail food market, was shown to be a sterol-requiring mollicute. Morphological examination of this organism by electron and dark-field microscopic techniques showed that it consists of small, nonhelical, nonmotile, pleomorphic coccoid cells, with individual cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew rapidly in all conventional culture medium formulations for mollicutes in either aerobic or anaerobic environments. The optimum temperature for growth was 30°C, but multiplication occurred at 18 to 37°C. Strain 831-C4T catabolized glucose, but hydrolysis of arginine or urea could not be demonstrated. The genome size of strain 831-C4T was determined to be about 569 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was 30.0 mol%. Recent studies in which we compared the 16S rRNA sequences of strain 831-C4T with those of more than 40 other mollicutes indicated that this organism is phylogenetically related to the Spiroplasma-Mycoplasma mycoides clade. Strain 831-C4T 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. Strain 831-C4T (= ATCC 49193) is the type strain of Mycoplasma lactucae sp. nov.

Three sterol-requiring mollicutes from floral surfaces of two tropical plant species (Melaleuca quinquenervia and Melaleuca decora) and a single isolate from a flower of the silk oak (Grevillea robusta) were serologically indistinguishable. Strain M1T (T = type strain), isolated from Melaleuca quinquenervia, was chosen for characterization. Light and electron microscopic observations of strain M1T revealed nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew well in SP-4 medium, but no sustained growth occurred in conventional mycoplasma media containing horse serum. The optimum temperature for growth was 23°C, but multiplication occurred over a temperature range of 10 to 30°C. Growth was not observed at temperatures above 30°C. Strain M1T and related strains (strains M5, M10, and SO1) catabolized glucose but hydrolyzed neither arginine nor urea. The size of the strain M1T genome was about 561 megadaltons, while the guanine-plus-cytosine content of the DNA was about 27.0 mol%. The organism was serologically unrelated to the type strains of the 80 previously recognized Mycoplasma species or to 18 other unclassified sterol-requiring strains cultivated from animal, plant, or insect sources. Recent sequencing studies of 16S rRNA demonstrated that strain M1T is a member of a clade that contains the type species of the genus Mycoplasma. Strain M1 (= ATCC 49191) is the type strain of Mycoplasma melaleucae sp. nov.

Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.

The taxonomic status of the anaerobe Bacteroides nodosus has for some time been uncertain. To resolve this uncertainty, the distal portion of a 16S rRNA gene from this important ovine pathogen was cloned, mapped, and sequenced. A comparison of the sequence with the sequences of 16S rRNA molecules from other bacteria indicated that B. nodosus is more closely related to Escherichia coli and other members of the class Proteobacteria than to Bacteroides fragilis or the bacteroides-flavobacterium-cytophaga phylum. The evidence from the comparison of sequence signatures suggests that B. nodosus is not a member of the genus Bacteroides but that it belongs in subgroup γ of the class Proteobacteria.

Strain PYAN-1T (T = type strain), which was isolated from a pupal gut of the firefly beetle Pyractonema angulata, and strains PIMN-1T and PIPN-2T, which were isolated from guts of adult Photinus marginalis and Photinus pyralis fireflies, respectively, were demonstrated to be sterol-requiring mollicutes. Cells of the three strains were shown by electron and dark-field microscopy to be small, pleomorphic, nonhelical, nonmotile bodies surrounded by single membranes. No evidence of a cell wall was observed, and the organisms were not susceptible to 500 U of penicillin per ml. The three strains grew rapidly in SP-4 broth medium. Strains PIMN-1T and PIPN-2T grew in medium supplemented with bovine serum fraction, but strain PYAN-1T did not. All three strains grew on solid media when the cultures were incubated aerobically, but only strains PYAN-1T and PIPN-2T formed colonies when anaerobic conditions were employed. The three strains catabolized glucose but hydrolyzed neither arginine nor urea. All of the strains grew at temperatures of 18 to 32°C; strains PYAN-1T and PIMN-1T also grew at 10°C. The optimal temperature for growth for strains PYAN-1T and PIPN-2T was 30°C; strain PIMN-1T grew equally well at 30 or 32°C. None of the three strains grew at 37°C. The genome sizes of strains PYAN-1T, PIMN-1T, and PIPN-2T were about 527 (478 to 589), 570 (480 to 630), and 762 (635 to 871) megadaltons, respectively. The guanine-plus-cytosine contents of the DNAs of the strains, as determined by buoyant density, thermal denaturation, and high-performance liquid chromatography methods, were as follows: 27.4, 30.2, and 27.5 mol%, respectively, for strain PYAN-1T; 28.8, 31.2, and 29.2 mol%, respectively, for strain PIMN-1T; and 28.4, 30.4, and 31.2 mol%, respectively, for strain PIPN-2T. All three strains were serologically unrelated to the type strains of previously established Mycoplasma species (including Mycoplasma ellychniae), to each other, and to 15 other unclassified sterol-requiring strains isolated from animals, plants, and insects. Along with M. ellychniae, these three organisms represent an unusual cluster of strains belonging to the genus Mycoplasma isolated from insects. Strain PYAN-1 (= ATCC 49194) is the type strain of Mycoplasma somnilux sp. nov.; strain PIMN-1 (= ATCC 49195) is the type strain of Mycoplasma luminosum sp. nov.; and strain PIPN-2 (= ATCC 49196) is the type strain of Mycoplasma lucivorax sp. nov.

Five moderately halophilic gram-positive cocci were isolated from soils and ponds of a solar saltern located near Alicante, Spain. These organisms all were nonmotile, nonsporing, reddish orange pigmented, strictly aerobic, and catalase and oxidase positive, had meso-diaminopimelic acid in their cell walls, and had DNA base compositions ranging from 45.7 to 49.3 mol%; they constitute a homology group with levels of DNA-DNA homology ranging from 70 to 100%. On the basis of their features, we regard these strains as belonging to a new species of the genus Marinococcus, for which we propose the name Marinococcus hispanicus. The type strain is strain J-82 (= ATCC 49259 = DSM 5352 = CCM 4148).

Nocardia nova strains were clearly distinguished from Nocardia asteroides, Nocardia farcinica, and other Nocardia species by numerical classification in which 107 characters were used. N. nova was differentiated from N. asteroides sensu stricto by the following characteristics: Presence of 2-week arylsulfatase, α-esterase, and β-esterase activities; absence of the ability to utilize citrate as a sole carbon source; and presence of the ability to utilize iso-butanol as a sole carbon source. The type strain (strain ATCC 33726) exhibited 39% relatedness to N. asteroides type strain ATCC 19247 and 20% relatedness to N. farcinica type strain ATCC 3318 in a DNA relatedness analysis. The α-mycolic acids of N. nova contained 52, 54, 56, 58, 60, and 62 carbon atoms and one to four double bonds. The numbers of carbon atoms in the α-unit were 16:0 or 18:1; these numbers in the α-unit were characteristic because the α-unit possessed an unsaturated alkyl unit (18:1). Thus, the mycolic acids of N. nova were clearly differentiated from those of N. asteroides and N. farcinica.

Eleven strains of a slow-growing, gram-negative bacterium causing corky root (CR) of lettuce were examined for morphological, physiological, and biochemical traits. Each strain consisted of small, motile rods with one lateral, subpolar, or polar flagellum. All strains were oligotrophic. Typical colonies were nonpigmented, umbonate, firm, and ultimately wrinkled. All strains were aerobic, metabolized glucose oxidatively, were oxidase positive and weakly catalase positive, and reduced nitrate to nitrite and ammonia but not to nitrogen gas. All of the strains tested were nitrogenase and arginine dihydrolase negative. Ethanol was not converted to acetic acid, and none of the 11 CR strains grew at pH 5.1 or below. The CR bacterium did not fluoresce and did accumulate poly-β-hydroxybutyrate granules. The CR strains did not hydrolyze starch or Tween 80, but did hydrolyze Tween 20. Very few carbon sources were utilized. The only isoprenoid quinone detected was ubiquinone Q10. The whole-cell fatty acid profiles of the CR strains consisted of several saturated and unsaturated straight-chain fatty acids, 2-hydroxy fatty acids, one cyclofatty acid, and one methylated fatty acid and resembled the fatty acid profile of Pseudomonas paucimobilis. The guanine-plus-cytosine content of the DNA was 59 mol%, which is below the range for P. paucimobilis. DNA-DNA homology studies indicated that the CR bacterium and P. paucimobilis are related but not the same species. The quinone and fatty acid compositions of the CR bacterium and P. paucimobilis differ substantially from those of other Pseudomonas spp., indicating that these organisms do not belong to the genus Pseudomonas proper. The characteristics of the CR bacterium do not conform to those of any previously described genus, and we propose a new genus, Rhizomonas, with one species, Rhizomonas suberifaciens, for strains of the CR bacterium. Additional tests will be needed to determine whether P. paucimobilis can be transferred to the genus Rhizomonas. Strain CA1 is the type strain of R. suberifaciens and has been deposited in the American Type Culture Collection and the National Collection of Plant Pathogenic Bacteria as strains ATCC 49355 and NCPPB 3629, respectively, together with strains FL1 (= ATCC 49356 = NCPPB 3628), NY11 (= ATCC 49382 = NCPPB 3631), and WI3 (=ATCC 49381 = NCPPB 3630). One additional strain that caused CR of lettuce had physiological and biochemical traits similar to those of R. suberifaciens but produced yellow colonies, had a higher guanine-plus-cytosine content (64 mol%), and exhibited lower DNA homology (54%) with DNA of the type strain. More strains will need to be studied to determine the taxonomic position of this strain.

Three Lactobacillus plantarum and seven Lactobacillus reuteri strains were studied by using restriction endonuclease analysis (REA) combined with principal-component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Chromosomal DNAs from the strains were extracted and cleaved with restriction enzymes, and the DNA fragments were separated according to size by agarose gel electrophoresis. Band patterns were read by using a laser densitometer. The procedure to obtain reproducible digestion patterns with a suitable number of DNA fragments was optimized. Asp 718, ClaI and EcoRI were the most suitable of the 17 restriction enzymes tested, each giving 30 to 50 DNA fragments down to a molecular weight of 4.2 × 106. The digestion patterns of all three enzymes, which gave a data set for 10 strains and 80 variables, were used for classification by PCA and SIMCA. All strains were clearly separated, and the separation within each species was in general accordance with data on DNA-DNA homology found in the literature. A class model was created for L. reuteri 1073, 1068, 1063, 1048, and 1044. Closest to this model was L. reuteri DSM 20016T (T = type strain), followed by L. reuteri DSM 20015; the most distant strains were the L. plantarum strains. We concluded that restriction endonuclease analysis, combined with PCA and SIMCA, can be used for the classification of Lactobacillus spp.

The generic position of a gram-positive, facultatively methylotrophic actinomycete known as Nocardia sp. strain 239 was determined by comparing reverse transcriptase sequences of 16S rRNA. The assignment of the organism to the genus Amycolatopsis was strongly supported by chemotaxonomic and morphological data. A comparison with the type strains of validly described Amycolatopsis species showed that the organism formed the nucleus of a new species. The name proposed for this new species is Amycolatopsis methanolica. The organism has been deposited in the National Collection of Industrial Bacteria as NCIB 11946.

It was recently proposed that the genus Bacteroides should be restricted to Bacteroides fragilis (the type species) and closely related organisms (viz., B. caccae, B. distasonis, B. eggerthii, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis, and B. vulgatus). By contrast, the moderately saccharolytic, predominantly oral Bacteroides species, which include B. melaninogenicus, B. oralis, and related species, form a phenotypically and phylogenetically coherent group of species which differ so significantly from the emended description of the genus Bacteroides that they should not be classified in the same genus. Therefore, we formally propose that these species be reclassified in a new genus, Prevotella. The type species is Prevotella melaninogenica.

An extremely halophilic red archaebacterium isolated from the Dead Sea (Ginzburg et al., J. Gen. Physiol. 55:187-207, 1970) belongs to the genus Haloarcula and differs sufficiently from the previously described species of the genus to be designated a new species; we propose the name Haloarcula marismortui (Volcani) sp. nov., nom. rev. because of the close resemblance of this organism to “Halobacterium marismortui,” which was first described by Volcani in 1940. The type strain is strain ATCC 43049.

The names Campylobacter laridis, Enterobacter intermedium, and “Flavobacterium branchiophila” are revised to Campylobacter lari, Enterobacter intermedius, and “Flavobacterium branchiophilum,” respectively, according to Rule 12c of the International Code of Nomenclature of Bacteria.