- Volume 55, Issue 4, 2005

Four strains of novel, rapidly growing, acid–alcohol-fast-staining bacteria were characterized with a polyphasic approach. Isolates were received by the Centers for Disease Control and Prevention from domestic health department laboratories for reference testing as unidentifiable, clinical mycobacteria. Bacteria were rod-shaped and produced non-pigmented (white to beige), non-photochromogenic, smooth or wrinkled-rough colonies on Middlebrook 7H10 and 7H11 media at 33 °C. The smooth and wrinkled colony forms were representative of two species with 68·0 and 72·0 mol% DNA G+C content. The cell wall contained meso-diaminopimelic acid and mycolic acids. Species were characterized by cellular fatty acids of C10 : 0, C14 : 0, C16 : 1ω9t, C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0 (tuberculostearic acid). HPLC analysis of mycolic acids produced a novel late-emerging, genus-specific mycolate pattern. TLC analysis demonstrated a novel α +-mycolate. Species were 98·9 % similar by comparison of 16S rRNA gene sequences; however, the DNA–DNA association was <28 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrated an association with Rhodococcus equi, although a DNA–DNA relatedness value of 2 % did not support a close relationship. PCR analysis of a proposed, selected actinomycete-specific 439 bp fragment of the 65 kDa heat-shock protein was negative for three of the four isolates. The creation of Segniliparaceae fam. nov. is proposed to encompass the genus Segniliparus gen. nov., including two novel species, the type species Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov., with the respective type strains CDC 1076T (=ATCC BAA-972T=CIP 108378T) and CDC 945T (=ATCC BAA-974T=CIP 108380T).

Fourteen strains isolated from vegetable sources and identified as belonging to Lactobacillus plantarum presented an atypical pattern of amplification with a species-specific multiplex-PCR assay. Phylogenetic analysis of two protein-encoding genes, recA (encoding the recombinase A protein) and cpn60 (encoding the GroEL chaperonin), as well as phenotypic and genomic traits revealed a homogeneous group of very closely related strains for which subspecies status is proposed, with the name Lactobacillus plantarum subsp. argentoratensis. The type strain is DKO 22T (=CIP 108320T=DSM 16365T).

The description of the Gram-positive, obligately parasitic, mycelial and endospore-forming bacterium, Pasteuria nishizawae, is emended to include additional observations on the life cycle, host specificity and endospore morphology. The nucleotide sequence of the 16S rRNA gene is also provided.

During the course of a 16S rRNA gene sequence phylogenetic evaluation of putative Glycomyces strains, it was noted that strain NRRL B-16338T is phylogenetically nearest to the genus Glycomyces but apparently is not a member of this or any of the other currently described actinomycete genera. The strain was subjected to a polyphasic study using standard methods for chemotaxonomic, morphological and physiological evaluation. The strain exhibited chemotaxonomic characteristics distinct from Glycomyces in spite of having 16S rRNA gene sequence similarity of 92 % with the described species of this genus. The whole-cell sugar pattern of NRRL B-16338T consisted of ribose and inositol, with traces of arabinose and mannose. The phospholipids observed were phosphatidylglycerol and diphosphatidylglycerol and menaquinones consisting of MK-10(H4), MK-10(H6), MK-11(H4) and MK-11(H6). A significant quantity (14·5 %) of 17 : 0 anteiso 2-hydroxy fatty acid was observed in the fatty acid profile of this strain. These characteristics clearly differentiate NRRL B-16338T from members of the genus Glycomyces and it is proposed that the strain represents a new genus within the family Glycomycetaceae to be called Stackebrandtia gen. nov. The description of this family is emended to permit its inclusion. It is proposed that the type species of the genus should be named Stackebrandtia nassauensis. The type strain LLR-40K-21T (=NRRL B-16338T=DSM 44728T) was isolated from a soil sample from Nassau, Bahamas.

A novel Gram-positive, aerobic, catalase-negative, coccus-shaped organism originating from tobacco was characterized using phenotypic and molecular taxonomic methods. The organism contained a cell wall murein based on l-lysine (variation A4α, type l-lysine–l-glutamic acid), synthesized long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types (with C16 : 1 ω9, C16 : 0 and C18 : 1 ω9 predominating) and possessed a DNA G+C content of 46 mol%. Based on morphological, biochemical and chemical characteristics, the coccus-shaped organism did not conform to any presently recognized taxon. Comparative 16S rRNA gene sequencing studies confirmed the distinctiveness of the unknown coccus, with the bacterium displaying sequence divergence values of greater than 7 % with other recognized Gram-positive taxa. Treeing analysis reinforced its distinctiveness, with the unidentified organism forming a relatively long subline branching at the periphery of an rRNA gene sequence cluster which encompasses the genera Alloiococcus, Allofustis, Alkalibacterium, Atopostipes, Dolosigranulum and Marinilactibacillus. Based on phenotypic and molecular phylogenetic evidence, it is proposed that the unknown organism from tobacco be classified as a new genus and species, Atopococcus tabaci gen. nov., sp. nov. The type strain of Atopococcus tabaci is CCUG 48253T (=CIP 108502T).

A Gram-positive, spore-forming, syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov. strain HHT, was isolated as a co-culture with Methanospirillum hungatei JF-1T from anaerobic, freeze-dried granular sludge obtained from an upflow anaerobic sludge bed reactor treating sugar beet wastewater. The bacterium converted propionate to acetate in co-culture with Methanospirillum hungatei JF-1T or Methanobacterium formicicum MFNT, but not in co-culture with Methanobrevibacter arboriphilus AZ. The organism could not be cultured axenically with any of the substrates tested and therefore can be considered as a (the first) true anaerobic syntrophic bacterium. The bacterium contained two distinct 16S rRNA gene sequences, with 96·8 % sequence similarity, which were both expressed during syntrophic growth on propionate as revealed by fluorescent in situ hybridization. The most closely related organisms are Cryptanaerobacter phenolicus LR7.2T, a bacterium that transforms phenol into benzoate, and Pelotomaculum thermopropionicum SIT, a thermophilic, syntrophic propionate-oxidizing bacterium. Other related species belong to the Gram-positive, sulfate-reducing genus Desulfotomaculum. The type strain of Pelotomaculum schinkii is strain HHT (=ATCC BAA-615T=DSM 15200T).

A bacterial strain (DB5T), with polysaccharide-degrading activities, was isolated from garden soil in Daejeon, Republic of Korea. The cells were Gram-positive, aerobic or facultatively anaerobic, non-motile straight rods. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas and that it is most closely related to Cellulomonas xylanilytica LMG 21723T and Cellulomonas humilata ATCC 25174T (98·0 and 97·9 % similarity, respectively). Chemotaxonomic data also supported the classification of strain DB5T in the genus Cellulomonas, i.e. l-ornithine as the cell-wall diamino acid, anteiso-C15 : 0 and iso-C15 : 0 as the major fatty acids, MK-9(H4) as the predominant menaquinone and the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol mannosides in the polar lipid profile. The results of DNA–DNA hybridization in combination with chemotaxonomic and physiological data demonstrated that strain DB5T (=KCTC 19081T=NBRC 100819T) should be classified as the type strain of a novel species within the genus Cellulomonas, for which the name Cellulomonas terrae sp. nov. is proposed.

An alkaliphilic, slightly halotolerant, chemo-organotrophic, Gram-positive, rod-shaped bacterium, strain 69B4T, was isolated from the sediment of the littoral zone of Lake Bogoria, Kenya. Phylogenetically, it is a member of the genus Cellulomonas, showing less than 97·5 % sequence similarity to the type strains of other Cellulomonas species. The highest level of similarity, albeit moderate, was found with respect to Cellulomonas cellasea DSM 20118T. Chemotaxonomic properties confirm the 16S rRNA gene-based generic affiliation, i.e. a DNA G+C content of 71·5 mol%, anteiso-C15 : 0 and C16 : 0 as the major fatty acids, MK-9(H4) as the major isoprenoid quinone, a peptidoglycan containing l-ornithine as the diamino acid and d-aspartic acid in the interpeptide bridge and phosphatidylglycerol as the only identified main polar lipid. The strain is aerobic to facultatively anaerobic, being capable of growth under strictly anaerobic conditions. Optimal growth occurs between pH values 9·0 and 10·0. On the basis of its distinct phylogenetic position and metabolic properties, strain 69B4T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas bogoriensis sp. nov. is proposed. The type strain is 69B4T (=DSM 16987T=CIP 108683T).

In order to clarify the taxonomic position of an actinobacterium from the Roman catacomb of Domitilla, a combination of phenotypic characterization, phylogenetic analysis based on the 16S rRNA gene sequence and DNA–DNA relatedness studies was used. The results from the polyphasic taxonomic study of this organism showed that strain HKI 0342T (=DSM 16849T=NCIMB 14033T) should be considered as the type strain of a novel species of the genus Isoptericola, for which the name Isoptericola hypogeus sp. nov. is proposed.

Six strains representing a novel yeast species belonging to the genus Saturnispora were isolated from two species of the Drosophila fasciola subgroup (Drosophila repleta group) in an Atlantic rainforest site in Rio de Janeiro State, Brazil. Four strains were isolated from crops and one from external parts of Drosophila cardinae. The other strain was isolated from external parts of Drosophila fascioloides. Analysis of the D1/D2 large-subunit rDNA sequences indicated that the novel species is closely related to Saturnispora dispora. The name Saturnispora hagleri sp. nov. is proposed to accommodate these strains. The type strain is UFMG-55T (=CBS 10007T=NRRL Y-27828T).

Twenty-one strains of a novel ascomycetous yeast species were isolated from soil collected in three kinds of natural forest, namely a dry dipterocarp forest, a mixed deciduous forest and a pine forest, in Nam Nao National Park, Phetchabun province, Thailand. The strains formed asci containing one to four ovoid to reniform ascospores, assimilated glucose, galactose and glycerol, fermented glucose and galactose vigorously and contained ubiquinone Q-6, indicating that they belonged to the genus Tetrapisispora. A comparative analysis of the small subunit rDNA (SSU rDNA) and the D1/D2 domain of the large subunit rDNA (LSU rDNA) of all available sequences for ascomycetous yeasts confirmed that the strains were phylogenetically related to the genus Tetrapisispora. All strains had identical nucleotide sequences in the D1/D2 domain of the LSU rDNA and differed from the nearest species, Tetrapisispora arboricola IFO 10925T, by 6·4 % nucleotide substitutions. The strains differed from Tetrapisispora arboricola by the ability to assimilate d-gluconic acid, the inability to grow on 50 % glucose medium, the nuclear DNA base composition and deliquescent asci. The strains were differentiated from the other four species of Tetrapisispora on the basis of trehalose assimilation, the ability to grow on 50 % glucose or 10 % NaCl plus 5 % glucose, vitamin requirement, the nuclear DNA base composition and the type of ascus. Based on the characteristics mentioned above, the strains are recognized as a single novel species of the genus Tetrapisispora and the name Tetrapisispora namnaonensis sp. nov. is proposed. The type strain is TN1-01T (=TISTR 5828T=JCM 12664T=CBS 10093T).

The nucleotide sequences (604 bp) of partial heat-shock protein genes (hsp65) from 161 Mycobacterium strains containing 56 reference Mycobacterium species and 105 clinical isolates were determined and compared. hsp65 sequence analysis showed a higher degree of divergence between Mycobacterium species than did 16S rRNA gene analysis. Generally, the topology of the phylogenetic tree based on the hsp65 DNA sequences was similar to that of the 16S rRNA gene, thus revealing natural relationships among Mycobacterium species. When a direct sequencing protocol targeting 422 bp sequences was applied to 70 non-tuberculous mycobacterium (NTM) clinical isolates, all NTMs were clearly identified. In addition, an XhoI PCR restriction fragment length polymorphism analysis method for the differentiation of Mycobacterium tuberculosis complex from NTM strains was developed during this study. The results obtained suggest that 604 bp hsp65 sequences are useful for the phylogenetic analysis and species identification of mycobacteria.

Unikaryotic enteromonads and diplokaryotic diplomonads have been regarded as closely related protozoan groups. It has been proposed that diplomonads originated within enteromonads in a single event of karyomastigont duplication. This paper presents the first study to address these questions using molecular phylogenetics. The sequences of the small-subunit rRNA genes for three isolates of enteromonads were determined and a tree constructed with available diplomonad, retortamonad and Carpediemonas sequences. The diplomonad sequences formed two main groups, with the genus Giardia on one side and the genera Spironucleus, Hexamita and Trepomonas on the other. The three enteromonad sequences formed a clade robustly situated within the diplomonads, a position inconsistent with the original evolutionary proposal. The topology of the tree indicates either that the diplokaryotic cell of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated by secondary reduction from the diplokaryotic state.