- Volume 55, Issue 1, 2005

The phylogeny of human isolates of Haemophilus species was estimated based on partial sequences of four separate housekeeping genes. The clustering of each set of sequences was in accordance with speciation of the strains with few exceptions: of 108 gene fragments examined, only three appeared to have been subject to recombination events across the species barrier. Housekeeping gene similarity supported previous DNA–DNA hybridization data for the genus rather than the phylogeny inferred from 16S rRNA gene sequence comparison. The similarity of sequences of Haemophilus parainfluenzae with those of Haemophilus influenzae suggested preservation of the former species in the genus Haemophilus. Three strains representing a novel taxon were unique with respect to the four investigated gene loci. 16S rRNA gene sequence analysis suggested that this taxon belonged to the Parainfluenzae cluster. DNA–DNA hybridization data supported this generic placement. Nine strains of the novel taxon were available for analysis. They were distinct from representatives of all current species of the genus Haemophilus by conventional phenotypic characterization. Genotypic and phenotypic data show that the strains merit recognition as a novel species of Haemophilus. The name Haemophilus pittmaniae sp. nov. is proposed, with HK 85T (=CCUG 48703T=NCTC 13334T) as the type strain.

A novel, thermophilic, obligately chemolithoautotrophic, sulfur/thiosulfate-oxidizing bacterium was isolated from subsurface geothermal aquifer water (temperature approximately 70 °C) in the Hishikari gold mine, Japan. Cells of the isolate, designated strain C55T, were motile, straight rods with a single polar flagellum. Growth was observed at temperatures between 35 and 62 °C (optimum 50–55 °C; 60 min doubling time) and pH between 5·2 and 7·7 (optimum pH 6·5–7·0). High growth rate of strain C55T was observed on either thiosulfate or elemental sulfur as a sole energy source, with molecular oxygen as the only electron acceptor. None of the organic compounds tested supported or stimulated growth of strain C55T. The G+C content of the genomic DNA was 66·9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C55T was affiliated to the β-Proteobacteria, but was distantly related to recognized genera. On the basis of its physiological and molecular properties, strain C55T (=JCM12421T=DSM 16629T=ATCC BAA-941T) is proposed as the type strain of Thiobacter subterraneus gen. nov., sp. nov.

Six strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from the Mediterranean Sea. 16S rRNA gene sequence analysis indicated that the strains were affiliated within the alphaproteobacterial genus Brevundimonas, with Brevundimonas intermedia (99·4 %) and Brevundimonas vesicularis (99·2 %) as their closest relatives. This affiliation was supported by chemotaxonomic data (major polar lipids: phosphatidyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidyl glucopyranosyl diacylglycerol; major fatty acids: C18 : 1, C16 : 0, C16 : 1, C15 : 0, C17 : 1 ω8c, 11-Me-C18 : 1 ω5t). The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strains from all recognized Brevundimonas species. The strains therefore represent a novel species, for which the name Brevundimonas mediterranea sp. nov. is proposed, with the type strain V4.BO.10T (=LMG 21911T=CIP 107934T).

Eight strains of Lactobacillus with identical partial 16S rRNA gene sequences and similar randomly amplified polymorphic DNA patterns were isolated from fermentation samples from Japanese and Scottish malt whisky distilleries. Phylogenetic analysis of almost complete 16S rRNA gene sequences from three representative strains (two from Japan, one from Scotland) placed them in the genus Lactobacillus as members of the Lactobacillus acidophilus group. Lactobacillus helveticus and Lactobacillus gallinarum were the most closely related species, with 16S rRNA gene similarities of 99·3 and 98·1 %, respectively. A similar phylogeny was derived from partial sequences of elongation factor Tu (tuf) genes in which the alleles from the three distillery isolates were identical and shared 99·0 % similarity with L. helveticus and L. gallinarum tuf genes. S-layer (slp) gene sequences suggested different relationships among the strains and the distillery isolates no longer formed a monophyletic group. The alleles from the Japanese and Scottish strains shared only 54 % similarity. Chromosomal DNA from the distillery strains gave DNA–DNA hybridization values between 79 and 100 % but showed less than 43 and 22 % reassociation with L. helveticus and L. gallinarum DNA, respectively. The name Lactobacillus suntoryeus sp. nov. is proposed for this novel taxon; the type strain is strain SAT (=LMG 22464T=NCIMB 14005T).

Screening of sourdough lactic acid bacteria for bacteriocin production resulted in the isolation of a Gram-positive, catalase-negative, non-spore-forming, non-motile rod bacterium (strain CS1T) that could not be associated with any previously described species. Comparative 16S rRNA gene sequence analysis recognized strain CS1T as a distinct member of the genus Lactobacillus. By a species-specific PCR strategy, five additional strains previously isolated from sourdoughs were found to belong to the same species as strain CS1T, as confirmed by 16S rRNA gene sequence analysis. The closest related species were Lactobacillus durianis, Lactobacillus malefermentans and Lactobacillus suebicus, with which strain CS1T shared 93 % sequence similarity. For a further characterization of strain CS1T, physiological (growth temperature, CO2 production, hydrolysis of arginine, isomeric type of lactate, sugar fermentation) and chemotaxonomic (G+C content and peptidoglycan structure) properties were determined. Phenotypic characterization showed that strain CS1T was a member of the obligately heterofermentative group of the genus Lactobacillus. The DNA G+C content was 44·6 mol%. The peptidoglycan was of the A3α (l-lys–l-ser–l-Ala2) type. Physiological, biochemical and genotypic data, as well as results of DNA–DNA hybridization of genomic DNA with one of the closest phylogenetic relatives, L. durianis (34·3 %), indicated that strain CS1T represents a novel species of the genus Lactobacillus for which the name Lactobacillus rossii sp. nov. is proposed. The type strain of this species is CS1T (=ATCC BAA-822T=DSM 15814T).

A Gram-positive short rod isolated from a saline soil in China was characterized using a polyphasic approach. This actinobacterium grew over a wide salinity range [0–15 % NaCl, 0–20 % KCl and 0–30 % MgCl2.6H2O (w/v); optimum concentrations for growth were 5 % NaCl, 5–10 % KCl, 10 % MgCl2.6H2O]. The optimum growth temperature and pH were 28–30 °C and 7·0–8·0, respectively. Chemotaxonomic features (peptidoglycan-type B2β with glycolyl residues; major menaquinones MK-10 and MK-11; predominating iso- and anteiso-branched cellular fatty acids; DNA G+C content 66·5 mol%) placed this organism within the genus Microbacterium. 16S rRNA gene sequence analysis confirmed this classification of the strain, but showed that it was distinct from its nearest neighbours. It formed a separate branch with type strains of the genus Microbacterium, and also shared low sequence similarity with them (<96 %). Based on phenotypic and molecular taxonomic results, it is proposed that the unknown isolate should be classified as a novel species in the genus Microbacterium, for which the name Microbacterium halotolerans sp. nov. is proposed. The type strain is YIM 70130T (=KCTC 19017T=CIP 108071T).

In an attempt to study the composition of the Lactobacillus flora from mucosa of human stomach, 16S rRNA gene sequences of 129 isolates were analysed. For 15 of these, the results differed significantly from known sequences, and additional tests were performed to determine whether these isolates represented as yet unrecognized species. Phenotypic and genetic characteristics revealed that these isolates represented four novel Lactobacillus species. Two belong to the Lactobacillus reuteri and the other two to the Lactobacillus delbrueckii subgroup of Lactobacillus. The names Lactobacillus gastricus sp. nov., Lactobacillus antri sp. nov., Lactobacillus kalixensis sp. nov. and Lactobacillus ultunensis sp. nov. are proposed, with the respective type strains Kx156A7T (=LMG 22113T=DSM 16045T=CCUG 48454T), Kx146A4T (=LMG 22111T=DSM 16041T=CCUG 48456T), Kx127A2T (=LMG 22115T=DSM 16043T=CCUG 48459T) and Kx146C1T (=LMG 22117T=DSM 16047T=CCUG 48460T).

Gram-positive, rod-shaped, motile lactic acid bacteria (strains NRIC 0603, NRIC 0604T, NRIC 0605 and NRIC 0606) were isolated from shochu mashes using an enrichment culture approach. These strains clustered in the Lactobacillus casei–Pediococcus group and were closely related to Lactobacillus nagelii and Lactobacillus mali on the basis of 16S rRNA gene sequence similarity. Levels of DNA–DNA relatedness revealed genotypic separation of the four isolates from the above two species. The isolates are therefore considered to represent a novel species, for which the name Lactobacillus satsumensis is proposed. The type strain is NRIC 0604T (=JCM 12392T=DSM 16230T).

An actinobacterium, strain YIM 70010T, which was isolated from a desert soil sample collected in Egypt, was subjected to a polyphasic taxonomy study. The organism was alkalitolerant and its optimum growth occurred at pH 8·0–9·0. The isolate contained chemotaxonomic markers that were characteristic of the genus Citricoccus, i.e. the peptidoglycan type Lys–Gly–Glu (variation A4α), the predominant menaquinone MK-9(H2) and a polar lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown glycolipids. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The G+C content of the genomic DNA was 63·8 mol%. Strain YIM 70010T exhibited a 16S rRNA gene sequence similarity of 99·6 % and DNA–DNA relatedness value of 56 % with Citricoccus muralis DSM 14442T. The phenotypic characteristics and DNA–DNA relatedness data indicate that strain YIM 70010T can be distinguished from C. muralis (DSM 14442T). Therefore, on the basis of the polyphasic taxonomic data presented, a novel species of the genus Citricoccus, Citricoccus alkalitolerans sp. nov. (type strain, YIM 70010T=CCTCC AA 203008T=DSM 15665T=KCTC 19012T) is proposed.

The taxonomic position of a bacterium isolated from a cold desert of the Himalayas, India, was analysed by using a polyphasic approach. The isolated strain, designated K22-03T, had phenotypic characteristics that matched those of the genus Planococcus and it represents a novel species. The almost-complete 16S rRNA gene sequence (1464 bases) of the novel strain was compared with those of previously studied Planococcus type strains and confirmed that the strain belongs to the genus Planococcus. 16S rRNA gene sequence analysis indicated that strain K22-03T differs from all other species of Planococcus by at least 2·5 %. DNA–DNA hybridization showed that it had low genomic relatedness with Planomicrobium mcmeekinii (MTCC 3704T, 23 %), Planococcus psychrophilus (MTCC 3812T, 61 %), Planococcus antarcticus (MTCC 3854T, 45 %) and Planomicrobium okeanokoites (MTCC 3703T, 51 %), the four species with which it was most closely related based on 16S rRNA gene sequence analysis (97–97·5 % similarity). Therefore, strain K22-03T should be recognized as a novel species, for which the name Planococcus stackebrandtii sp. nov. is proposed. The type strain is K22-03T (=MTCC 6226T=DSM 16419T=JCM 12481T).

Two Gram-positive, rod-shaped, spore-forming and moderately halophilic bacteria (strains 28-1T, 28-4), isolated from a soil sample from a neutral salt lake in Xin-Jiang, China, were characterized polyphasically. On the basis of fasta (ungapped) analyses of 16S rRNA gene sequences, strains 28-1T and 28-4 were shown to belong to the Bacillaceae and to be closely related to Filobacillus milensis DSM 13259T (97·0 %) and Bacillus haloalkaliphilus DSM 5271T (95·7 %). 16S rRNA gene sequence similarity with other recognized species was not more than 94·1 %. Phylogenetic, chemotaxonomic, physiological and biochemical data supported the differentiation of these novel strains from F. milensis and B. haloalkaliphilus. Therefore these two previously unidentified strains are considered to represent a new genus and species, for which the name Tenuibacillus multivorans gen. nov., sp. nov. is proposed. The type strain is 28-1T (=AS 1.3442T=NBRC 100370T).

A group of nine Gram-positive endospore-forming bacteria was isolated from soil of the Drentse A agricultural research area in the Netherlands. Using (GTG)5-PCR genomic fingerprinting and fatty acid analysis, the nine isolates were divided into three consistent groups. On the basis of 16S rRNA gene sequence similarity of representative strains, the nine isolates were shown to belong to the genus Bacillus. The first group of four isolates was most closely related to Bacillus carboniphilus (95·5 %) and Bacillus sporothermodurans (95·5 %). The second and third groups of three and two isolates, respectively, showed highest sequence similarity to Bacillus neidei (97·0 and 97·1 %, respectively) and Bacillus pycnus (both 96·7 %). A DNA–DNA relatedness study confirmed the consistency of the three groups delineated by (GTG)5-PCR and fatty acid analysis. A small number of phenotypic characters allowed differentiation of the three groups of isolates. The three groups therefore represent novel species, for which the names Bacillus humi, Bacillus arenosi and Bacillus arvi are proposed, with LMG 22167T (=DSM 16318T), LMG 22166T (=DSM 16319T) and LMG 22165T (=DSM 16317T) as the respective type strains.

A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen). (GTG)5-PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups. Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species ‘Bacillus macroides’ (strain NCIMB 8796) and ‘Bacillus maroccanus’ (names not validly published) according to a fasta search. The close similarity between B. simplex, ‘B. macroides’ NCIMB 8796, ‘B. maroccanus’ and the mural painting isolates was confirmed by additional (GTG)5-PCR, ARDRA, FAME and SDS-PAGE analyses. Furthermore, these techniques revealed that strains of ‘Bacillus carotarum’, another name that has not been validly published, also showed high similarity to this group of organisms. On the other hand, it was shown that the strains labelled ‘B. macroides’ in different collections do not all belong to the same species. Strain NCIMB 8796 can be allocated to B. simplex, while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98·6 %). On the basis of further DNA–DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus, for which the name Bacillus muralis sp. nov. is proposed. Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to ‘B. macroides’ and strains belonging to ‘B. maroccanus’ and ‘B. carotarum’ are allocated to the species B. simplex and an emended description of B. simplex is given.

A Gram-positive actinobacterium, previously classified as Kocuria varians, was subjected to a polyphasic taxonomic study. The bacterium showed the peptidoglycan type Lys–Ala3 (variation A3α), MK-7(H2) was the major menaquinone and anteiso-C15 : 0 and anteiso-C17 : 0 were the major fatty acids. On the basis of the phylogenetic and phenotypic characteristics of the actinobacterium, a novel species, Kocuria carniphila sp. nov. (type strain, CCM 132T=DSM 16004T), is proposed.

A polyphasic study was carried out to clarify the taxonomic position of two Gram-positive bacteria isolated from soil samples of the Grotta dei Cervi (Italy), a relatively unexplored hypogean environment. The strains, 20-5T and 23-23T, showed phenotypic and phylogenetic characteristics that were consistent with their classification in the genus Agromyces. 16S rRNA gene sequence comparisons revealed that the two strains formed distinct phyletic lines within the genus Agromyces. Based on 16S rRNA gene sequence similarity, chemotaxonomic data and the results of DNA–DNA relatedness studies, it is proposed that the two isolates represent two novel species of the genus Agromyces. Pronounced differences in a broad range of phenotypic characteristics and DNA G+C content distinguished the two strains from each other and from previously described species of the genus Agromyces. Two novel species are proposed: Agromyces salentinus sp. nov. (type strain, 20-5T=HKI 0320T=DSM 16198T=NCIMB 13990T) and Agromyces neolithicus sp. nov. (type strain, 23-23T=HKI 0321T=DSM 16197T=NCIMB 13989T).

A haloalkaliphilic Gram-positive bacterium, strain 6AGT, that grew aerobically at an optimum temperature of 37 °C and at pH 7–10 (optimum 9·0), was isolated from algal mat from a mineral pool located in Malvizza in the Campania region (southern Italy). The isolate tolerated high concentrations of NaCl, up to 25 %. On the basis of 16S rRNA gene sequence similarity, the strain was shown to belong to the genus Bacillus. Analysis of the 16S rRNA gene sequence revealed high similarity between strain 6AGT and an unidentified isolate from Hailaer soda lake (China) (99·9 % identity) and two Kenyan isolates, 3E1 and WE4 (98·3 and 97·8 % identity, respectively). The G+C content of the DNA was 48·4 mol%. The predominant respiratory quinones were MK-7(H2), MK-7(H4) and DMK-7(H2); phosphatidylglycerol and diphosphatidylglycerol were the predominant polar lipids. iC15 : 0 and aiC15 : 0 were the major fatty acids. Strain 6AGT accumulated osmolytes. The phylogenetic distance of strain 6AGT (=DSM 15402T=ATCC BAA-957T) from any recognized species within the genus Bacillus allowed it to be classified as the type strain of Bacillus saliphilus sp. nov.

Three moderately halophilic, spore-forming strains, designated BH030062T, BH030049 and BH030080, were isolated from a solar saltern in Korea. Phylogenetic analyses and comparative 16S rRNA gene sequence studies revealed that the isolates represent a novel distinct monophyletic lineage within the phyletic group classically defined as the genus Bacillus and are most closely related to members of the genera Gracilibacillus (93·7–95·1 % similarity), Virgibacillus (93·5–94·8 %), Halobacillus (94·8–95·9 %), Filobacillus (94·4–94·8 %) and Lentibacillus (93·3–93·7 %). Strain BH030062T was strictly aerobic, rod-shaped, Gram-positive and motile by means of peritrichous flagella. It grew in the presence of 1–15 % (w/v) NaCl and at temperatures of 15–45 °C. The cell wall peptidoglycan contained A1γ-meso-diaminopimelic acid as the diagnostic diamino acid. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. DNA G+C content was about 41 mol% and the major isoprenoid quinone was MK-7. On the basis of their physiological and molecular properties, the isolates represent a new genus, Pontibacillus gen. nov., and novel species, Pontibacillus chungwhensis sp. nov. The type strain is BH030062T (=KCTC 3890T=DSM 16287T).

Two Gram-positive, endospore-forming bacterial strains, CR-502T and CR-14b, which produce surfactant molecules are described. Phenotypic tests and phylogenetic analyses showed these strains to be members of the genus Bacillus and related to the species Bacillus atrophaeus, Bacillus mojavensis, Bacillus subtilis, Bacillus vallismortis and Bacillus amyloliquefaciens, although they differ from these species in a number of phenotypic characteristics. DNA–DNA hybridization confirmed that they show less than 20 % hybridization with the above-mentioned species and therefore represent a novel species of Bacillus. The DNA G+C content is 46·4 mol% in strain CR-502T and 46·1 mol% in strain CR-14b. The main fatty acids in strain CR-502T are 15 : 0 anteiso (32·70 %), 15 : 0 iso (29·86 %) and 16 : 0 (13·41 %). The main quinone in strain CR-502T is MK-7 (96·6 %). In the light of the polyphasic evidence gathered in this study, it is proposed that these strains be classified as a novel species of the genus Bacillus, with the name Bacillus velezensis sp. nov. The type strain (CR-502T=CECT 5686T=LMG 22478T) was isolated from a brackish water sample taken from the river Vélez at Torredelmar in Málaga, southern Spain.

An anaerobic bacterium that transforms phenol and 4-hydroxybenzoate (4-OHB) into benzoate, strain LR7.2T, was isolated from a culture originating from a mixture of swamp water, sewage sludge, swine waste and soil. Cells of strain LR7.2T are Gram-positive short rods (1×2 μm) that are electron-dense when observed by electron microscopy. The optimum pH and temperature for growth and transformation activity of 4-OHB are 7·5–8·0 and 30–37 °C, respectively. The bacterium does not use sulphate, thiosulphate, nitrate, nitrite, FeCl3, fumarate or arsenate as an electron acceptor. It does not normally use sulphite, although stimulation of growth and 4-OHB transformation activity at a low concentration (up to 2 mM) has been reported previously under different culture conditions. The presence of 4-OHB or phenol is essential for growth; transformation of 4-OHB or phenol into benzoate is used to produce energy for growth. Using [6D]-phenol, 4-OHB was shown to be an intermediate in the transformation of phenol into benzoate. No spore was observed. The bacterium has a DNA G+C content of 51 mol% and its major membrane fatty acid is anteiso-C15 : 0. The 16S rRNA gene sequence of strain LR7.2T shows only 90 % similarity to its closest relative (Pelotomaculum thermopropionicum). From these results, a new taxon is proposed: Cryptanaerobacter phenolicus gen. nov., sp. nov. The type strain is LR7.2T (=ATCC BAA-820T=DSM 15808T).