- Volume 55, Issue 5, 2005

Four thermophilic, Gram-positive strains, designated H0165T, 500275T, C0170 and 700375, were isolated from a composting process in Japan. The isolates grew aerobically at about 65 °C on a solid medium with formation of substrate mycelia; spores were produced singly along the mycelia. These morphological characters resembled those of some type strains of species belonging to the family ‘Thermoactinomycetaceae’, except that aerial mycelia were not formed. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the closest related species to the isolates were members of the family ‘Thermoactinomycetaceae’, but that the isolates formed an independent phylogenetic lineage. Some chemotaxonomic characters of the isolates, such as DNA G+C contents of 58·7–60·3 mol%, MK-7 as the major menaquinone and cellular fatty acid profiles, differed from those of members of the family ‘Thermoactinomycetaceae’. DNA–DNA hybridization showed that the isolates could be divided into two genomic groups, strain H0165T and the other three strains. These results indicated that the four isolates should be classified into two species of a novel genus in the family ‘Thermoactinomycetaceae’, for which the names Planifilum fimeticola gen. nov., sp. nov. (type strain H0165T=ATCC BAA-969T=JCM 12507T) and Planifilum fulgidum sp. nov. (type strain 500275T=ATCC BAA-970T=JCM 12508T) are proposed.

Two bacterial strains, designated D-1,5aT and D-1,5b, were isolated from a medieval wall painting in the chapel of Castle Herberstein, Styria (Austria). The Gram-positive, heterotrophic, aerobic, spore-forming rods showed nearly identical whole-cell protein patterns, identical genomic fingerprints and identical physiological profiles, demonstrating their relationship at the species level. Both strains contained meso-diaminopimelic acid in their peptidoglycan, possessed a quinone system comprising menaquinone MK-7 and had fatty acid profiles in which C15 : 0 iso and C15 : 0 anteiso were predominant. The 16S rRNA gene sequence of D-1,5aT showed the highest similarity (99·5 %) to the sequence of Bacillus sp. LMG 20243, and Bacillus flexus IFO 15715T was the next most closely related established species (96·5 %). Other type strains, such as Bacillus fastidiosus DSM 91T, Bacillus indicus SD/3T, Bacillus cibi JG-30T, Bacillus megaterium IAM 13418T, Bacillus cohnii DSM 6308T, Bacillus bataviensis LMG 21833T and Bacillus soli LMG 21838T, shared 96·0–96·1 % 16S rRNA gene sequence similarity with D-1,5aT. The combination of physiological and chemotaxonomic traits distinguishes the two strains from those species sharing the highest sequence similarities (96·0–96·5 %). On the basis of these characteristics and the phylogenetic position of strain D-1,5aT (=DSM 16534T=CCM 7228T), this strain is assigned as the type strain of a novel species of the genus Bacillus, for which the name Bacillus herbersteinensis sp. nov. is proposed.

A mesophilic, acidophilic, spore-forming bacterium, strain 105-2T, was isolated from an acidic Sphagnum peat bog in Wisconsin, USA. Strain 105-2T has 16S rRNA gene sequence similarity to Bacillus sporothermodurans DSM 10599T and Bacillus oleronius DSM 9356T of 97·4 and 97·8 %, respectively. The primary lipoquinone is MK-7 and the major fatty acids are 15 : 0 iso, 15 : 0 anteiso and 17 : 0 anteiso. The predominant polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and a glycolipid. The DNA G+C content was found to be 43·2 mol%. The phenotypic, chemotaxonomic and molecular analyses identified strain 105-2T as a novel Bacillus species, for which the name Bacillus acidicola is proposed. The type strain is 105-2T (=DSM 14745T=ATCC BAA-366T=NRRL B-23453T).

A moderately thermophilic, anaerobic, chemolithoheterotrophic, sulfate-reducing bacterium, strain CO-1-SRBT, was isolated from sludge from an anaerobic bioreactor treating paper mill wastewater. Cells were Gram-positive, motile, spore-forming rods. The temperature range for growth was 30–68 °C, with an optimum at 55 °C. The NaCl concentration range for growth was 0–17 g l−1; there was no change in growth rate until the NaCl concentration reached 8 g l−1. The pH range for growth was 6·0–8·0, with an optimum of 6·8–7·2. The bacterium could grow with 100 % CO in the gas phase. With sulfate, CO was converted to H2 and CO2 and part of the H2 was used for sulfate reduction; without sulfate, CO was completely converted to H2 and CO2. With sulfate, strain CO-1-SRBT utilized H2/CO2, pyruvate, glucose, fructose, maltose, lactate, serine, alanine, ethanol and glycerol. The strain fermented pyruvate, lactate, glucose and fructose. Yeast extract was necessary for growth. Sulfate, thiosulfate and sulfite were used as electron acceptors, whereas elemental sulfur and nitrate were not. A phylogenetic analysis of 16S rRNA gene sequences placed strain CO-1-SRBT in the genus Desulfotomaculum, closely resembling Desulfotomaculum nigrificans DSM 574T and Desulfotomaculum sp. RHT-3 (99 and 100 % similarity, respectively). However, the latter strains were completely inhibited above 20 and 50 % CO in the gas phase, respectively, and were unable to ferment CO, lactate or glucose in the absence of sulfate. DNA–DNA hybridization of strain CO-1-SRBT with D. nigrificans and Desulfotomaculum sp. RHT-3 showed 53 and 60 % relatedness, respectively. On the basis of phylogenetic and physiological features, it is suggested that strain CO-1-SRBT represents a novel species within the genus Desulfotomaculum, for which the name Desulfotomaculum carboxydivorans is proposed. This is the first description of a sulfate-reducing micro-organism that is capable of growth under an atmosphere of pure CO with and without sulfate. The type strain is CO-1-SRBT (=DSM 14880T=VKM B-2319T).

Two novel Pediococcus strains, Z-1 and Z-8T, were isolated from a distilled-spirit-fermenting cellar in Hebei Province, China. The cells were Gram-positive, non-spore-forming, non-motile cocci, occurring in pairs or tetrads. The strains were facultatively anaerobic and produced 50 % (−)-d-/50 % (+)-l-lactic acid exclusively from glucose fermentation, but did not produce catalase. Both strains fermented rhamnose, ribose, xylose, lactose, maltose, trehalose and sucrose, and tolerated 10 % ethanol. The mean G+C content of the two strains was 38±1 mol% (37 mol% for the type strain, Z-8T). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Z-8T clustered within the Pediococcus damnosus–Pediococcus inopinatus–Pediococcus parvulus group, with 98·3–98·5 % 16S rRNA gene similarity. DNA–DNA relatedness between strain Z-8T and P. damnosus LMG 11484T, P. inopinatus LMG 11409T and P. parvulus LMG 11486T was 19±4, 35±5 and 31±5 %, respectively. Therefore, based on the distinctive phenotypic characteristics, and genotypic and phylogenetic analyses, a novel Pediococcus species, Pediococcus cellicola sp. nov., is proposed. The type strain is Z-8T (=AS 1.3787T=LMG 22956T).

A Gram-positive, aerobic, non-motile bacterial strain, KSL-107T, was isolated from an alkaline soil from Korea and subjected to a polyphasic taxonomic study. Strain KSL-107T grew optimally at 25 °C and pH 7·0–7·5. Strain KSL-107T had a cell-wall peptidoglycan based on ll-2,6-diaminopimelic acid. It contained MK-9(H4) as the predominant menaquinone and C16 : 0, C16 : 0 2-OH and 10-methyl C18 : 0 as the major fatty acids. The DNA G+C content was 71·5 mol%. Comparative 16S rRNA gene sequence analyses showed that strain KSL-107T was phylogenetically affiliated to the genus Aeromicrobium of the family Nocardioidaceae. Similarity values between the 16S rRNA gene sequence of strain KSL-107T and those of type strains of Aeromicrobium species were in the range 97·9–98·2 %. The isolate could be distinguished from other Aeromicrobium species by levels of DNA–DNA relatedness and differences in some phenotypic characteristics. On the basis of the data presented, it is suggested that strain KSL-107T (=KCTC 19073T=DSM 16824T) represents a novel Aeromicrobium species, for which the name Aeromicrobium alkaliterrae sp. nov. is proposed. An emended description of the genus Aeromicrobium is also proposed.

The taxonomic position of strain LMG 13590T, originally isolated from dog faeces and classified as Enterococcus dispar in the BCCM/LMG Bacteria Catalogue, was reinvestigated. This strain and 12 recent isolates from faecal samples of healthy dogs occupied a clearly separate position when investigated with multilocus sequence analysis (MLSA) of the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS). The 16S rRNA gene sequence of one representative strain showed highest similarities of 98–99 % with E. dispar LMG 13521T, Enterococcus canis LMG 12316T and Enterococcus asini LMG 18727T. A further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA–DNA hybridization and biochemical features demonstrated that the 13 enterococcal dog faecal strains represent a single, novel Enterococcus species for which the name Enterococcus canintestini sp. nov. is proposed. The type strain is LMG 13590T (=CCM 7285T).

Two enterococcal strains LMG 16607T and LMG 16612 originating from sea water were analysed in a polyphasic taxonomic study. Both strains, assigned as Enterococcus sp. in the BCCM/LMG culture collection, possessed analogous protein profiles, but these were different from all other enterococcal species. 16S rRNA gene sequence analysis of one strain showed the highest similarity, 96·9–96·1 %, with its closest phylogenetic neighbours Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus saccharominimus and Enterococcus italicus. Further genomic analysis by (GTG)5-PCR fingerprinting and sequence analysis of the housekeeping gene phenylalanyl-tRNA synthase (pheS) and distinct biochemical features confirmed that the two strains represent a novel enterococcal species for which the name Enterococcus aquimarinus sp. nov. is proposed. The type strain is LMG 16607T (=CCM 7283T).

Lactobacillus ferintoshensis has recently been described as a novel species, distinct from its close phylogenetic neighbours Lactobacillus buchneri, Lactobacillus kefiri and Lactobacillus hilgardii. Two highly related species with validly published names, Lactobacillus parakefiri and Lactobacillus parabuchneri, were not considered in the study due to the lack of 16S rRNA gene sequence data at that time. Since the publication of the study, the sequences have become available and have revealed that L. ferintoshensis and L. parabuchneri share 99·7 % 16S rRNA gene sequence similarity. Further genomic and phenotypic data, derived from fluorescent amplified fragment length polymorphism, DNA–DNA hybridization and API 50 CHL analyses, have demonstrated that the species are synonymous.

Two novel Lactobacillus strains, C-5-1T and HB5, were isolated from the walls of a distilled spirit fermenting cellar in Hebei province, China. The strains were Gram-positive, non-spore-forming, non-motile curved rods and were facultatively anaerobic. They produced 5 % (−)-d/95 % (+)-l lactic acid exclusively from glucose fermentation, but were catalase-negative. Both strains fermented N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, trehalose and β-gentiobiose. The mean DNA G+C content of the two strains was 46·9±0·8 mol% (46·4 mol% for the type strain, C-5-1T). Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain C-5-1T was clustered in the Lactobacillus casei/Pediococcus phylogenetic group and was closely related to Pediococcus dextrinicus JCM 5887T (97·9 % similarity), followed by Lactobacillus algidus JCM 10491T (93·9 % similarity). The DNA–DNA relatedness between the type strain C-5-1T and P. dextrinicus JCM 5887T and L. algidus JCM 10491T was only 5·4 and 4·3 %, respectively. Therefore, based on phenotypic, genotypic and phylogenetic analyses, a novel Lactobacillus species, Lactobacillus concavus sp. nov., is proposed. The type strain is C-5-1T (=AS 1.5017T=LMG 22739T).

A Gram-positive, rod- or coccus-shaped bacterial strain, KSL-104T, was isolated from an alkaline soil from Korea and its taxonomic position was investigated by a polyphasic approach. Strain KSL-104T grew optimally at pH 7·0–8·0 and 30 °C. It was characterized chemotaxonomically as having a cell-wall peptidoglycan type based on ll-2,6-diaminopimelic acid with MK-8(H4) as the predominant menaquinone. The major fatty acid was iso-C16 : 0 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The DNA G+C content was 70·6 mol%. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain KSL-104T joined the cluster comprising Nocardioides jensenii and Marmoricola aurantiacus. The cellular fatty acid profile of strain KSL-104T was different from that of M. aurantiacus. Strain KSL-104T and the type strain of N. jensenii exhibited a 16S rRNA gene sequence similarity value of 97·1 % and a mean DNA–DNA relatedness value of 13 %. Levels of 16S rRNA gene sequence similarity between strain KSL-104T and the type strains of other Nocardioides species were in the range 94·0–96·5 %. On the basis of phenotypic, phylogenetic and genetic data, strain KSL-104T (=KCTC 9992T=JCM 13008T) represents a novel species of the genus Nocardioides, for which the name Nocardioides dubius sp. nov. is proposed.

Strains belonging to two novel yeast species, Candida bromeliacearum and Candida ubatubensis, were isolated from the bromeliad tank of Canistropsis seidelii (Bromeliaceae) in a sandy coastal plain (restinga) ecosystem site in an Atlantic rainforest of south-eastern Brazil. These species were genetically distinct from all other currently accepted ascomycetous yeasts, based on sequence divergence in the D1/D2 domains of the large-subunit rDNA and in the small-subunit rDNA. The species occupy basal positions in the Metschnikowiaceae clade. The type strains are Candida bromeliacearum UNESP 00-103T (=CBS 10002T=NRRL Y-27811T) and Candida ubatubensis UNESP 01-247RT (=CBS 10003T=NRRL Y-27812T).

The unidentified strains AS 2.0706T, preserved in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, and CBS 6904T, preserved in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, were shown to represent two novel ascomycetous yeast species of the genus Kazachstania by 18S rDNA, internal transcribed spacer (ITS) region (including 5·8S rDNA) and 26S rDNA D1/D2 domain sequence analysis and electrophoretic karyotype comparison. The names Kazachstania aquatica sp. nov. and Kazachstania solicola sp. nov. are proposed for strains AS 2.0706T and CBS 6904T, respectively. Phylogenetically, the two novel species are closely related to Kazachstania aerobia, Kazachstania servazzii and Kazachstania unispora.

Three strains (N002, N069 and PT31T) of a novel thermotolerant methylotrophic yeast species belonging to the genus Pichia were isolated from soil collected in Thailand by three consecutive enrichments in methanol broth at room temperature. They were categorized as thermotolerant strains on the basis of their good growth below 20 °C and up to a high temperature (37 °C). The major characteristics of the three strains included the following and placed them in the genus Pichia: the formation of four helmet-/hat-shaped ascospores in a deliquescent ascus that might be unconjugated or produced by conjugation between a cell and its bud or between independent cells; multilateral budding; the presence of ubiquinone Q-7; negative for Diazonium blue B colour and urease reactions; and the absence of arthrospores and ballistospores. The three strains differed by one to three nucleotide substitutions in the sequences of the D1/D2 domain of the large-subunit rDNA sequence. Phylogenetic analysis revealed that their closest species was Pichia dorogensis, but with 11–13 nucleotide substitutions in 554 nt. The phenotypic characteristics of the three strains were the same. The strains could be distinguished from P. dorogensis by a number of phenotypic characteristics. On the basis of the above findings, these three strains were assigned to a single novel species of Pichia, for which the name Pichia thermomethanolica sp. nov. is proposed. The type strain is PT31T (=BCC 16875T=JCM 12984T=CBS 10098T).

The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase β-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

The rnpB gene is ubiquitous in Bacteria, Archaea and Eucarya and encodes the RNA component of RNase P, an endoribonuclease P that consists of one RNA and one protein subunit (C5). In this study, partial rnpB genes were sequenced from 39 type strains and 16 additional strains of the genus Legionella. Models of the putative secondary structures of the RNase P RNA in the genus Legionella are proposed and possible interactions between RNase P RNA and C5 are discussed. The phylogenetic relationships within the genus Legionella were examined and rnpB sequences indicated six main clades that together comprised 27 of the 39 species examined. The phylogenetic relationships were further inferred by analysing combined datasets of sequences from the rnpB, mip, 16S rRNA and rpoB genes. It is concluded that rnpB is suitable for use in phylogenetic studies of closely related species and that it exhibits the potential to discriminate between Legionella species.

Phytopathogenic mollicutes, which include spiroplasmas and phytoplasmas, are cell wall-less bacteria that parasitize plant hosts and insect vectors. Knowledge of the evolution of these agents is important in understanding their biology. The availability of the first complete phytoplasma and several partial spiroplasma and phytoplasma genome sequences made possible an investigation of evolutionary relationships between phytopathogenic mollicutes and other micro-organisms, especially Gram-positive bacteria, using a comparative genomics approach. Genome data from a total of 41 bacterial species were used in the analysis. Sixty-one conserved proteins were selected from each species for the construction of a hypothetical phylogenetic tree. The genes encoding these selected proteins are among a core of genetic elements that constitute a hypothetical minimal genome. The proteins were concatenated into five superproteins according to their functional categories, and phylogenetic trees were reconstructed using distance, parsimony and likelihood methods. Phylogenetic trees based on the five sets of concatenated proteins were congruent in both clade topology and relative branching length. Spiroplasma kunkelii and phytoplasmas clustered together with other mollicutes, forming a monophyletic group. Phytoplasmas diverged from spiroplasmas and mycoplasmas at early stages in the evolution of mollicutes. Branch lengths on the phylogenetic trees were noticeably longer in the Mollicutes clade, suggesting that the genes encoding the five sets of proteins evolved at a greater rate in this clade than in other clades. This observation reinforces the concept that mollicutes have rapidly evolving genomes.