- Volume 54, Issue 6, 2004

Four isolates, which were obtained from Belgian, Moroccan and Romanian dairy products, constituted a homogeneous but unidentified taxon after screening with whole-cell protein fingerprinting. Complete 16S rRNA gene sequence analysis classified representative strains in the genus Enterococcus. Highest sequence similarities of 98·6 and 98·0 % were obtained with the species Enterococcus sulfureus and Enterococcus saccharolyticus, respectively. Growth characteristics, biochemical features, tRNA intergenic length polymorphism analysis, DNA–DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel Enterococcus species. It differs phenotypically from other enterococci in characteristics commonly considered as typical of this genus: no growth in 6·5 % NaCl or 0·4 % sodium azide, and no acid production from a wide range of carbohydrates. The name Enterococcus saccharominimus sp. nov. is proposed for this novel species; the type strain (LMG 21727T=CCM 7220T) was isolated from contaminated pasteurized cow's milk.

A coryneform strain isolated from soda lake mud in China corresponded in chemotaxonomic characteristics such as peptidoglycan type A4α l-lys–l-ser–d-Glu and major menaquinone MK-9, as well as in its DNA base composition (57 mol% G+C), to its phylogenetic neighbour Jonesia denitrificans. Differences in phenotypic characteristics and the phylogenetic distance (96·6 % 16S rRNA gene sequence similarity) from J. denitrificans justify the proposal of a second species of the genus Jonesia, Jonesia quinghaiensis sp. nov., with the type strain QH3A7T (=DSM 15701T=CGMCC 1.3459T).

Eight unidentified Gram-positive, rod-shaped organisms were recovered from the tracheas of apparently healthy black storks (Ciconia nigra) and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria the isolates were tentatively assigned to the genus Corynebacterium, although three of the organisms did not appear to correspond to any recognized species. Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates were phylogenetically members of the genus Corynebacterium. Five strains were genotypically identified as representing Corynebacterium falsenii, whereas the remaining three strains represented a hitherto unknown subline, associated with a small subcluster of species that includes Corynebacterium mastitidis and its close relatives. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from black storks represent a novel species within the genus Corynebacterium, for which the Corynebacterium ciconiae sp. nov. is proposed. The type strain is CECT 5779T (=BS13T=CCUG 47525T).

Several aerobic, motile, rod-shaped, thermophilic, spore-forming Geobacillus bacteria predominantly giving a Gram-positive staining reaction were isolated from a cool soil environment in Northern Ireland and taxonomically investigated. Two isolates, F10 and TfT, showed low 16S rRNA gene sequence similarity to recognized members of the genus Geobacillus. Phylogenetic tree investigation using neighbour-joining, maximum-likelihood and parsimony methods indicated that strains F10 and TfT represent a single novel species, for which the name Geobacillus debilis sp. nov. is proposed, with type strain TfT (=DSM 16016T=NCIMB 13995T) and which belongs to a subgroup of the genus Geobacillus comprising Geobacillus toebii and Geobacillus caldoxylosilyticus. However, G. debilis showed closest affinities to Bacillus pallidus, which we propose should become Geobacillus pallidus comb. nov.

A polyphasic taxonomic approach was applied to strains of the species Staphylococcus vitulinus and Staphylococcus pulvereri in order to clarify their taxonomic relatedness. Four reference strains, representing both species, and seven strains isolated from human clinical material were characterized by biotyping, ribotyping and SDS-PAGE analysis of whole-cell proteins, and none of the screening approaches allowed the two taxa to be distinguished. DNA–DNA hybridization experiments between four selected representative strains, including the type strains, confirmed that Staphylococcus pulvereri is a later synonym of Staphylococcus vitulinus.

A strictly anaerobic arsenate-respiring bacterium isolated from a gold mine in Bendigo, Victoria, Australia, belonging to the genus Bacillus is described. Cells are Gram-positive, motile rods capable of respiring with arsenate and nitrate as terminal electron acceptors using a variety of substrates, including acetate as the electron donor. Reduction of arsenate to arsenite is catalysed by a membrane-bound arsenate reductase that displays activity over a broad pH range. Synthesis of the enzyme is regulated; maximal activity is obtained when the organism is grown with arsenate as the terminal electron acceptor and no activity is detectable when it is grown with nitrate. Mass of the catalytic subunit was determined to be approximately 87 kDa based on ingel activity stains. The closest phylogenetic relative, based on 16S rRNA gene sequence analysis, is Bacillus arseniciselenatis, but DNA–DNA hybridization experiments clearly show that strain JMM-4T represents a novel Bacillus species, for which the name Bacillus macyae sp. nov. is proposed. The type strain is JMM-4T (=DSM 16346T=JCM 12340T).

Two mesophilic anaerobic bacterial strains (Z7T and Z1) were isolated from waste water sludge of the Xinanzhang paper mill, Beijing, China. The strains were Gram-positive, non-spore-forming and motile. Cells were thin rods (0·2–0·4×4·0–8·0 μm). Growth of the strains was observed at 20–42 °C and pH 5·0–7·5. Both strains hydrolysed gelatin and aesculin and fermented several kinds of mono-, di- and oligosaccharides. The fermentation end products formed from glucose were acetate, ethanol, hydrogen and carbon dioxide. The predominant cellular fatty acids were the branched-chain fatty acids isoC15 : 0 (42·83 %) and isoC14 : 0 (32·11 %). The DNA G+C contents of strains Z7T and Z1 were 50·4 and 48·6 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates represent a new phyletic sublineage within the Clostridium leptum rRNA cluster, with <91 % 16S rRNA gene sequence similarity to currently described species. On the basis of polyphasic evidence from this study, Acetanaerobacterium elongatum gen. nov., sp. nov., a novel genus and species, is proposed, with strain Z7T (=JCM 12359T=AS 1.5012T) as the type strain.

Six isolates of an unknown Gram-positive, catalase-negative, chain-forming, coccus-shaped organism isolated from ovine and caprine mastitis were characterized by phenotypic and molecular taxonomic methods. On the basis of cellular morphology and the results of biochemical tests, the organism was tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed that the organism is a member of the genus Streptococcus, with Streptococcus equi as its closest phylogenetic relative (98·8 % similarity). DNA–DNA pairing studies showed that the unidentified organism displayed more than 70 % relatedness to the type strains of S. equi subsp. equi and subsp. zooepidemicus. Despite the relatively high DNA–DNA reassociation values, biotyping and ribotyping allowed clear differentiation of the unknown bacterium from the two recognized subspecies of S. equi. On the basis of phenotypic and molecular genetic evidence, it is proposed that the unknown Streptococcus isolates from ovine and caprine mastitis be classified as a novel subspecies, Streptococcus equi subsp. ruminatorum subsp. nov. The type strain is CECT 5772T (=CCUG 47520T=Mt 167T).

The taxonomic position of a soil isolate, strain 276T, was established using a polyphasic approach. The organism showed a range of chemical and morphological properties consistent with its classification in the genus Nocardia. An almost complete 16S rRNA gene sequence determined for the strain was aligned with corresponding sequences of representatives of the genus Nocardia and related taxa using three tree-making algorithms. The organism formed a distinct phyletic line within the evolutionary radiation occupied by the genus Nocardia and was most closely related to the type strain of Nocardia abscessus. However, the two strains shared a low DNA–DNA relatedness value and were readily distinguished using a combination of phenotypic properties. The combined genotypic and phenotypic data show that strain 276T should be assigned to the genus Nocardia as a novel species. The name proposed for this new taxon is Nocardia xishanensis sp. nov. The type strain is 276T (=CGMCC 4.1860T=JCM 12160T).

The taxonomic position of a polycyclic-aromatic-hydrocarbon-degrading bacterium, strain 17A3T, isolated from contaminated soil was determined using a combination of phenotypic and genotypic properties. The isolate showed phenotypic properties that were diagnostic for species of the genus Mycobacterium. Comparative 16S rRNA gene sequence analysis assigned 17A3T to the 16S rRNA gene subgroup that contains Mycobacterium aurum, Mycobacterium austroafricanum, Mycobacterium vaccae and Mycobacterium vanbaalenii, but it could clearly be distinguished from these species using a combination of physiological, chemotaxonomic markers and internal rRNA gene spacer analyses. The data showed that strain 17A3T (=DSM 44605T=NRRL B-24244T) merits recognition as the type strain of a novel species of the genus Mycobacterium. The name Mycobacterium pyrenivorans sp. nov. is proposed for the species because of its ability to use pyrene as a sole source of carbon and energy.

A polyphasic taxonomic evaluation of presumptive strains representative of the genus Glycomyces held within the Agricultural Research Service Culture Collection resulted in the discovery of three novel species. Analysis of the whole cell sugar content of these novel species, as well as of two species presently assigned to the genus, revealed that the whole cell sugar pattern was different from that reported in the formal description of the genus Glycomyces. The sugars present in all strains studied included ribose, xylose, mannose and galactose rather than xylose and arabinose as reported in the original description of the genus. Moreover, the menaquinone patterns observed for the novel species also deviated from the original genus description. The formal description of the genus Glycomyces is emended to reflect these new data. The novel species proposed and described are Glycomyces algeriensis sp. nov. (type strain NRRL B-16327T=DSM 44727T), Glycomyces arizonensis sp. nov. (type strain NRRL B-16153T=DSM 44726T) and Glycomyces lechevalierae sp. nov. (type strain NRRL B-16149T=DSM 44724T).

A new anaerobic, thermophilic, facultatively carboxydotrophic bacterium, strain Nor1T, was isolated from a hot spring at Norris Basin, Yellowstone National Park. Cells of strain Nor1T were curved motile rods with a length of 2·6–3 μm, a width of about 0·5 μm and lateral flagellation. The cell wall structure was of the Gram-negative type. Strain Nor1T was thermophilic (temperature range for growth was 40–68 °C, with an optimum at 60 °C) and neutrophilic (pH range for growth was 6·5–7·6, with an optimum at 6·8–7·0). It grew chemolithotrophically on CO (generation time, 1·15 h), producing equimolar quantities of H2 and CO2 according to the equation CO+H2O→CO2+H2. During growth on CO in the presence of ferric citrate or amorphous ferric iron oxide, strain Nor1T reduced ferric iron but produced H2 and CO2 at a ratio close to 1 : 1, and growth stimulation was slight. Growth on CO in the presence of sodium selenite was accompanied by precipitation of elemental selenium. Elemental sulfur, thiosulfate, sulfate and nitrate did not stimulate growth of strain Nor1T on CO and none of these chemicals was reduced. Strain Nor1T was able to grow on glucose, sucrose, lactose, arabinose, maltose, fructose, xylose and pyruvate, but not on cellobiose, galactose, peptone, yeast extract, lactate, acetate, formate, ethanol, methanol or sodium citrate. During glucose fermentation, acetate, H2 and CO2 were produced. Thiosulfate was found to enhance the growth rate and cell yield of strain Nor1T when it was grown on glucose, sucrose or lactose; in this case, acetate, H2S and CO2 were produced. In the presence of thiosulfate or ferric iron, strain Nor1T was also able to grow on yeast extract. Lactate, acetate, formate and H2 were not utilized either in the absence or in the presence of ferric iron, thiosulfate, sulfate, sulfite, elemental sulfur or nitrate. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. The DNA G+C content of the strain was 51·7±1 mol%. Analysis of the 16S rRNA gene sequence revealed that strain Nor1T belongs to the Bacillus–Clostridium phylum of the Gram-positive bacteria. On the basis of the studied phenotypic and phylogenetic features, we propose that strain Nor1T be assigned to a new genus, Thermosinus gen. nov. The type species is Thermosinus carboxydivorans sp. nov. (type strain, Nor1T=DSM 14886T=VKM B-2281T).

Two moderately thermophilic, Gram-positive, spore-forming bacteria were isolated from different geographical locations and sources; strain GS5-97T from a beet sugar factory in Leopoldsdorf, Lower Austria, and strain YNP10 from a geothermally heated soil, Yellowstone National Park, USA. The sequences of their 16S rRNA genes were found to be 99·8 % identical, and DNA–DNA hybridization experiments revealed that strains GS5-97T and YNP10 share 89·9 mol% similarity to each other, but only 34·3 and 39·2 mol% similarity, respectively, to Geobacillus caldoxylosilyticus DSM 12041T, which is their closest related type strain. A polyphasic analysis showed that these two isolates were more similar to each other than to other characterized geobacilli. Their DNA G+C content was 43·2 and 42·4 mol%, respectively, and they were identical with respect to many phenotypic features (e.g. Topt 55 °C; pHopt 7·0). Both strains clearly displayed best growth when cultured aerobically. They differed slightly in their cellular fatty acid profiles and polar lipid pattern, and genotypically they could also be distinguished based on randomly amplified polymorphic DNA fingerprints and internal transcribed spacer analysis. Freeze-etching experiments revealed oblique surface layer (S-layer) lattices in both strains, and biochemical analyses of the purified S-layer proteins indicated the occurrence of glycosylation. Based on the properties of these organisms relative to those currently documented for the genus Geobacillus and for the various sister genera in the Bacillus radiation, a novel species is proposed, Geobacillus tepidamans sp. nov., with GS5-97T (=ATCC BAA-942T=DSM 16325T) as the type strain. Strain YNP10 has been deposited in the American Type Culture Collection as ATCC BAA-943.

Two coryneform bacteria were isolated from a penguin rookery soil sample collected in Antarctica, near the Indian station Dakshin Gangotri (strain Lz1yT), and from sea water from Kerguelen island, Antarctica (strain KGN15T). They have morphological and chemotaxonomic properties (peptidoglycan A4α type; major menaquinones MK-8, MK-9 and MK-10; predominant fatty acids anteiso-C15 : 0 and anteiso-C17 : 0) that are characteristic of members of the genus Arthrobacter. The isolates shared 97·8 % 16S rRNA gene sequence similarity to each other and were most closely related to Arthrobacter sulfureus (about 98·5 % sequence similarity). DNA–DNA hybridization experiments revealed 50 % relatedness between the isolates, while the levels of DNA–DNA relatedness between strains Lz1yT and KGN15 T and their phylogenetic relative, A. sulfureus, were respectively 54 and 12 %. Based on the above data and distinct phenotypic differences between the isolates and A. sulfureus, two novel species are proposed, Arthrobacter gangotriensis sp. nov. (type strain Lz1yT=DSM 15796T=JCM 12166T) and Arthrobacter kerguelensis sp. nov. (type strain KGN15T=DSM 15797T=JCM 12165T).

A psychrotolerant, obligately alkaliphilic bacterium, IDR2-2T, which is able to reduce indigo dye was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) produced in Date, Hokkaido, using a traditional Japanese method. The isolate grew at pH 9–12 but not at pH 7–8. It was a Gram-positive, facultatively anaerobic, straight rod-shaped bacterium with peritrichous flagella. The isolate grew in 0–17 % (w/v) NaCl but not at NaCl concentrations higher than 18 % (w/v). Its major cellular fatty acids were C14 : 0, C16 : 0, C16 : 19c and C18 : 19c, and its DNA G+C content was 40·6 mol%. dl-lactic acid was the major end-product from d-glucose. No quinones could be detected. The peptidoglycan type was A4β, Orn–d-Glu. A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain IDR2-2T is a member of the genus Alkalibacterium. DNA–DNA hybridization revealed low relatedness (less than 25 %) between the isolate and two phylogenetically related strains, Alkalibacterium olivapovliticus and Marinilactibacillus psychrotolerans. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate merits classification as a novel species, for which the name Alkalibacterium psychrotolerans sp. nov. is proposed. The type strain is IDR2-2T (=JCM 12281T=NCIMB 13981T).

Four isolates of a rapidly growing Mycobacterium species had a mycolic acid pattern similar to that of Mycobacterium smegmatis, as determined by HPLC analyses. Three of the isolates were from footbath drains and a sink at a nail salon located in Atlanta, GA, USA; the fourth was obtained from a granulomatous subdermal lesion of a female patient in Venezuela who was undergoing mesotherapy. By random amplified polymorphic DNA electrophoresis and PFGE of large restriction fragments, the three isolates from the nail salon were shown to be the same strain but different from the strain from the patient in Venezuela. Polymorphisms in regions of the rpoB, hsp65 and 16S rRNA genes that were shown to be useful for species identification matched for the two strains but were different from those of other Mycobacterium species. The 16S rRNA gene sequence placed the strains in a taxonomic group along with Mycobacterium frederiksbergense, Mycobacterium hodleri, Mycobacterium diernhoferi and Mycobacterium neoaurum. The strains produced a pale-yellow pigment when grown in the dark at the optimal temperature of 35 °C. Biochemical testing showed that the strains were positive for iron uptake, nitrate reduction and utilization of d-mannitol, d-xylose, iso-myo-inositol, l-arabinose, citrate and d-trehalose. The strains were negative for d-sorbitol utilization and production of niacin and 3-day arylsulfatase, although arylsulfatase activity was observed after 14 days. The isolates grew on MacConkey agar without crystal violet but not on media containing 5 % (w/v) NaCl or at 45 °C. They were susceptible to ciprofloxacin, amikacin, tobramycin, cefoxitin, clarithromycin, doxycycline, sulfamethoxazole and imipenem. The name Mycobacterium cosmeticum sp. nov. is proposed for this novel species; two strains, LTA-388T (=ATCC BAA-878T=CIP 108170T) (the type strain) and 2003-11-06 (=ATCC BAA-879=CIP 108169) have been designated, respectively, for the strains of the patient in Venezuela and from the nail salon in Atlanta, GA, USA.

This study presents the first 18S rRNA multi-library environmental PCR survey of a single protozoan phylum, Cercozoa Cavalier-Smith 1998, from a range of different habitats. Phylogenetic analysis reveals at least nine novel clades within the phylum, several possibly at the level of order or above. Further experiments are described to ascertain the true ecological and geographical distributions of some clades that might be inferred from the tree to be restricted in either or both ways. These results suggest that the diversity of cercozoan taxa may run into thousands of lineages, making it comparable in diversity to the largest better-characterized protozoan phyla, e.g. Ciliophora (ciliates and suctorians) and Foraminifera. New sequences of cultured Spongomonas, Metromonas and Metopion are also presented. In the light of these additions, and the increased taxon sampling from the environmental libraries, some revisions of cercozoan classification are made: the transfer of Spongomonadea from Reticulofilosa to Monadofilosa; the removal of Metopiida from Sarcomonadea; and the creation of the new order Metromonadida, currently containing the single genus Metromonas. Although Metromonas groups with weak to moderate support with Chlorarachnea, it is here placed in superclass Monadofilosa, to which it is morphologically more similar.

The novel yeast species Candida leandrae is described based on eight isolates from decaying fruits of Leandra reversa Cogn. (Melastomataceae) in an Atlantic rainforest site in Brazil, one from a Convolvulaceae flower in Costa Rica and one from a drosophilid in Hawai'i. The strains differed in their colony morphology, one being butyrous and smooth and the other being filamentous and rugose. Sequences of the D1/D2 domains of the large-subunit rRNA gene from both morphotypes were identical. C. leandrae belongs to the Kodamaea clade and is closely related to Candida restingae. The two species can be separated on the basis of growth at 37 °C and the assimilation of melezitose, negative in the novel species. The type culture of C. leandrae is strain UNESP 00-64RT (=CBS 9735T=NRRL Y-27757T).

A major clade of new yeast taxa from the digestive tract of basidiocarp-feeding beetles is recognized based on rRNA gene sequence analyses. Almost 30 % of 650 gut isolates formed a statistically well-supported clade that included Candida tanzawaensis. The yeasts in the clade were isolated from 11 families of beetles, of which Tenebrionidae and Erotylidae were most commonly sampled. Repeated isolation of certain yeasts from the same beetle species at different times and places indicated strong host associations. Sexual reproduction was never observed in the yeasts. Based on comparisons of small- and large-subunit rRNA gene sequences and morphological and physiological traits, the yeasts were placed in Candida ambrosiae and in 16 other undescribed taxa. In this report, the novel species in the genus Candida are described and their relationships with other taxa in the Saccharomycetes are discussed. The novel species and their type strains are as follows: Candida guaymorum (NRRL Y-27568T=CBS 9823T), Candida bokatorum (NRRL Y-27571T=CBS 9824T), Candida kunorum (NRRL Y-27580T=CBS 9825T), Candida terraborum (NRRL Y-27573T=CBS 9826T), Candida emberorum (NRRL Y-27606T=CBS 9827T), Candida wounanorum (NRRL Y-27574T=CBS 9828T), Candida yuchorum (NRRL Y-27569T=CBS 9829T), Candida chickasaworum (NRRL Y-27566T=CBS 9830T), Candida choctaworum (NRRL Y-27584T=CBS 9831T), Candida bolitotheri (NRRL Y-27587T=CBS 9832T), Candida atakaporum (NRRL Y-27570T=CBS 9833T), Candida panamericana (NRRL Y-27567T=CBS 9834T), Candida bribrorum (NRRL Y-27572T=CBS 9835T), Candida maxii (NRRL Y-27588T=CBS 9836T), Candida anneliseae (NRRL Y-27563T=CBS 9837T) and Candida taliae (NRRL Y-27589T=CBS 9838T).