- Volume 53, Issue 2, 2003

Ramlibacter gen. nov. is proposed for two aerobic, chemo-organotrophic, cyst-producing soil bacterial strains. These bacteria are Gram-negative, non-flagellated rods or cysts, isolated from subdesert soil in Tataouine, Tunisia. Phylogenetic analyses of the rrs sequences of the two strains showed that they do not constitute a robust clade at the genus level with any previously described bacteria and that they are a deep branch of a clade also grouping the genera Acidovorax and Hydrogenophaga within the β-Proteobacteria. They belong to two different species, as verified by DNA–DNA hybridization (23·5 % reassociation). The type species of the genus is Ramlibacter tataouinensis sp. nov., with the type strain TTB310T (=DSM 14655T =ATCC BAA-407T =LMG 21543T). The second species is Ramlibacter henchirensis sp. nov., with the type strain TMB834T (=DSM 14656T =ATCC BAA-408T =LMG 21542T). The G+C contents of R. tataouinensis and R. henchirensis are 69·6 and 66·6 mol%, respectively.

Slowly growing, non-pigmented mycobacteria were isolated from striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. Growth characteristics, acid-fastness and results of 16S rRNA gene sequencing were consistent with those of the genus Mycobacterium. A unique profile of biochemical reactions was observed among the 21 isolates. A single cluster of eight peaks identified by analysis of mycolic acids (HPLC) resembled those of reference patterns but differed in peak elution times from profiles of reference species of the Mycobacterium tuberculosis complex. One isolate (M175T) was placed within the slowly growing mycobacteria by analysis of aligned 16S rRNA gene sequences and was proximate in phylogeny to Mycobacterium ulcerans and Mycobacterium marinum. However, distinct nucleotide differences were detected in the 16S rRNA gene sequence among M175T, M. ulcerans and M. marinum (99·2 % similarity). Isolate M175T could be differentiated from other slowly growing, non-pigmented mycobacteria by its inability to grow at 37 °C, production of niacin and urease, absence of nitrate reductase and resistance to isoniazid (1 µg ml−1), thiacetazone and thiophene-2-carboxylic hydrazide. Based upon these genetic and phenotypic differences, isolate M175T (=ATCC 700981T =NCTC 13215T) is proposed as the type strain of a novel species, Mycobacterium shottsii sp. nov.

Endospore-forming bacteria were isolated from insect-pathogenic nematodes, Heterorhabditis spp., from three diverse geographical locations. Spindle-shaped sporangia of these bacteria adhere to the free-living infective stage of the nematode, which carries them to new insect hosts, where the bacterium reproduces. These isolates were characterized based on phenotypic and chemotaxonomic properties and 16S rRNA gene sequences. Analysis of the 16S rRNA gene placed the isolates within the genus Paenibacillus. The isolates shared higher sequence similarities with each other (95·1–100 %) than they did with any other named species within the genus (89·2–94 %). Paenibacillus macquariensis, Paenibacillus azoreducens, Paenibacillus amylolyticus and Paenibacillus durus were among the species with highest sequence similarity to these isolates. The isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C15 : 0 and C16 : 0 were among the major fatty acid types and the DNA G+C content was approximately 44 mol% in all isolates. DNA–DNA similarity studies revealed genomic heterogeneity among the isolates, such that they are likely to represent more than one species. Two of the isolates (both from a Heterorhabditis megidis isolate from Estonia) are phenotypically distinguishable from the others and are proposed as a single species, Paenibacillus nematophilus sp. nov. The type strain for this novel species is NEM1aT (=DSM 13559T=NCIMB 13845T). The other isolates, although closely related to the proposed species, are likely to represent at least one, but most likely two, novel species.

Microbial growths causing discoloration on the Roman wall paintings of the Servilia tomb at the necropolis of Carmona (Spain) and the medieval wall paintings of the Saint-Catherine chapel at Castle Herberstein (Austria) were investigated and from four different samples, a group of ten strains with similar characteristics was isolated. The isolates were characterized in a polyphasic taxonomic study, including 16S rDNA sequence analysis, (GTG)5-PCR genomic fingerprinting, DNA–DNA hybridization, DNA base ratio, fatty acid analysis, morphological and biochemical characterization. The data obtained attribute the isolates to a novel species of the genus Bacillus, for which the name Bacillus decolorationis sp. nov. is proposed. The type strain is strain LMG 19507T (=DSM 14890T).

A novel bacterium, strain B33D1T, isolated from agricultural soil, was characterized taxonomically and phylogenetically. Strain B33D1T was a Gram-positive, aerobic rod of medium length that formed long chains on a common laboratory medium. However, B33D1T grew poorly on the surface of agar plates and was sensitive to desiccation. The optimal growth temperature was 30 °C (range 19–38 °C). The organism grew well on a variety of sugars and was capable of utilizing a few amino acids as sole carbon sources. Phylogenetically, the most closely related described species to strain B33D1T was Rubrobacter xylanophilus, which possessed 86 % 16S rRNA sequence similarity. However, a number of 16S rRNA gene clones derived from soil samples possessed up to 93 % sequence similarity. These results placed strain B33D1T within the subclass Rubrobacteridae of the phylum Actinobacteria. The novel genus and species Solirubrobacter pauli gen. nov., sp. nov. is proposed, with strain B33D1T (=ATCC BAA-492T=DSM 14954T) as the type strain.

A group of 13 strains was isolated from samples of biofilm formation on the mural paintings of the Servilia tomb (necropolis of Carmona, Spain) and the Saint-Catherine chapel (castle at Herberstein, Austria). The strains were subjected to a polyphasic taxonomic study, including (GTG)5-PCR, 16S rDNA sequence analysis, DNA–DNA hybridizations, DNA base ratio determination, analysis of fatty acids, polar lipids and menaquinones and morphological and biochemical characterization. In a phylogenetic tree based on neighbour-joining of 16S rDNA sequences, the strains are divided in two major groups, representing three novel species according to DNA–DNA relatedness, that are positioned at approximately equal distances from Virgibacillus and Salibacillus. After comparison of the novel results with existing data, the transfer of the species of Salibacillus to Virgibacillus is proposed, with the resulting new combinations Virgibacillus marismortui comb. nov. and Virgibacillus salexigens comb. nov. Additionally, three novel species are described, for which the names Virgibacillus carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus picturae sp. nov. are proposed. The respective type strains are LMG 20964T (=DSM 14868T), LMG 19488T (=DSM 14866T) and LMG 19492T (=DSM 14867T). Finally, an emended description of the genus Virgibacillus is given.

Lactobacillus versmoldensis sp. nov. (KU-3T) was isolated from raw fermented sausages. The new species was present in high numbers, and frequently dominated the lactic acid bacteria (LAB) populations of the products. 16S rDNA sequence data revealed that the isolates are closely related to the species Lactobacillus kimchii DSM 13961T, Lactobacillus paralimentarius DSM 13238T, Lactobacillus alimentarius DSM 20249T and Lactobacillus farciminis DSM 20184T. DNA–DNA reassociation data, however, clearly distinguished the new isolates from these species; they showed a low degree of DNA relatedness with the type strains of this group of phylogenetically closely related lactobacilli. These results warrant separate species status for strain KU-3T, for which the name Lactobacillus versmoldensis sp. nov. is proposed. The type strain is KU-3T (=DSM 14857T =NCCB 100034T =ATCC BAA-478T).

Two unknown, Gram-negative, catalase-negative and strictly anaerobic cocci were isolated from two independent human samples (strains AIP 49.01 and AIP 412.00T). Comparative 16S rRNA gene sequencing demonstrated that these two organisms displayed 99·8 % sequence identity and that they are members of the Sporomusa sub-branch of the low-G+C Gram-positive bacteria. The most closely related 16S rDNA sequences were from Megasphaera sp. oral clone BU057 (99·8 %) and from isolates of Megasphaera cerevisiae and Megasphaera elsdenii (94·5 and 93·8 %, respectively). Phylogenetic analysis based on 16S rDNA sequences showed that these two strains were most closely related to M. elsdenii and belonged to the Megasphaera genus. Differences from previously described Megasphaera species in terms of size, biochemical tests (particularly the analysis of metabolic end products), gas production and DNA G+C content indicated that the two strains studied represent a novel species of anaerobic Gram-negative cocci. The name Megasphaera micronuciformis sp. nov. is proposed for these two isolates. It is also proposed that the uncultured organism previously deposited as Megasphaera sp. oral clone BU057 should be named ‘Candidatus Megasphaera micronuciformis’. The type strain of Megasphaera micronuciformis is AIP 412.00T (=CIP 107280T =CCUG 45952T).

A novel Gram-positive bacterial strain was isolated from forest soil. According to its 16S rRNA sequence, this strain is a deep-rooting member of the class Actinobacteria. The 16S rRNA sequence is most closely related (∼94 % identity) to clones of uncultured bacteria detected in different terrestrial environments, while showing only a remote relationship (∼90 % identity or less) to sequences of cultured species. Cells of the first cultured representative of this phylogenetic cluster are small, short rods that are motile by peritrichous flagella, catalase- and oxidase-positive and grow under aerobic conditions. In liquid culture, flagella from different cells can aggregate to form networks, clearly visible under the light microscope. The peptidoglycan contains meso-diaminopimelic acid and is directly cross-linked (type A1γ). Mycolic acids are not present. The polar lipids are phosphatidylinositol and an unidentified phospholipid. Menaquinone MK-7(H4) was detected as the predominant isoprenoid quinone. Oleic, 14-methylpentadecanoic, hexadecanoic and ω6c-heptadecenoic acids are the predominant components of the cellular fatty acid profile. The DNA G+C content is 71 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of a new genus and species, Conexibacter woesei gen. nov., sp. nov., with the type strain ID131577T (=DSM 14684T =JCM 11494T).

Two Gram-positive, non-motile, non-spore-forming, halotolerant and moderately halophilic cocci (strains YKJ-101T and YKJ-115T) were isolated from the traditional Korean fermented seafood jeotgal, and were investigated using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rDNA sequences showed that strains YKJ-101T and YKJ-115T are most closely related to the cluster comprising two Salinicoccus species. The peptidoglycan type of the strains is A3α, based on l-Lys–Gly3–4–l-Ala(Gly), and the predominant menaquinone is MK-7. Strains YKJ-101T and YKJ-115T have cellular fatty acid profiles containing major amounts of saturated, unsaturated and branched fatty acids; the major fatty acids are anteiso-C15 : 0 and iso-C15 : 0. The cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol and unidentified phospholipids. Strains YKJ-101T and YKJ-115T have identical DNA G+C contents of 42 mol%. The 16S rDNA similarity between strains YKJ-101T and YKJ-115T is 98 % and the mean level of DNA–DNA relatedness between the two strains is 13·4 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, it is proposed that strains YKJ-101T and YKJ-115T should be placed in a new genus, Jeotgalicoccus gen. nov., as two distinct new species, for which the names Jeotgalicoccus halotolerans sp. nov. and Jeotgalicoccus psychrophilus sp. nov. are proposed. The type strains are YKJ-101T (=KCCM 41448T =JCM 11198T) and YKJ-115T (=KCCM 41449T =JCM 11199T), respectively.

A previously undescribed Actinomyces-like bacterium was isolated from a lesion in the jaw of a cow. Based on its cellular morphology and the results of biochemical testing, the organism was tentatively identified as a member of the genus Actinomyces. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto unknown species within the genus Actinomyces, and is related to a group of species that includes Actinomyces turicensis and its close relatives. It is proposed that the unknown organism be classified as Actinomyces vaccimaxillae sp. nov. (the type strain is CCUG 46091T=CIP 107423T).

Nuclear-encoded SSU rRNA genes from nine strains of Distigma and three strains of Astasia were sequenced and analysed phylogenetically with maximum-likelihood and maximum-parsimony methods. It could be demonstrated that the genus Distigma is paraphyletic, consisting of two distinct clades: one comprises four strains of the type species, Distigma proteus, and the other includes four strains of Distigma curvatum, Distigma gracile, Distigma sennii and Distigma elegans. These findings are well corroborated by morphological characteristics. The investigated species of Astasia are closely related to members of the Rhabdomonadida, thus rendering the genus Astasia polyphyletic, with Astasia longa branching within the phototrophs. All of the species investigated cluster in a well-supported group of primary osmotrophic euglenids that are not derived from photosynthetic ancestors. The recovered clades are characterized by their sequence diversity. After different evolutionary rates among lineages had been determined, a modified slow–fast approach was used to differentiate phylogenetic signal from noise. Finally, a revised systematic scheme based on phylogenetic relationships is suggested to render euglenid taxonomy more transparent: primary osmotrophic euglenids are classified as Aphagea, and members of the D. curvatum group are transferred into the new subgenus Parvonema.

The term ‘flexispira’ refers to micro-organisms with a particular morphology: fusiform-shaped with helical periplasmic fibrils and bipolar tufts of sheathed flagella. Two flexispira taxa have been formally named, Helicobacter bilis and Helicobacter trogontum, a third named species is Helicobacter aurati and eight additional 16S rRNA sequence-based flexispira taxa have been described by Dewhirst et al. (Int J Syst Evol Microbiol 50, 1781–1787, 2000) and given the provisional designation Helicobacter sp. flexispira taxa 1–5, 7, 8 and 10. In the present study, seven gastric or intestinal flexispira isolates from seven Finnish pigs originating from different farms were characterized. Morphologically, all these porcine isolates had typical flexispira morphology. Analysis of the 16S rDNA sequences of five isolates showed that they were most closely related to the sequences of flexispira taxa 4 and 5 and H. trogontum (taxon 6), but less closely related to taxa 1–3 and 8, H. bilis and H. aurati. Phenotypic characterization, analysis of RFLPs of 16S and 23S rDNAs and SDS-PAGE profiles revealed that all of the porcine isolates, reference strains of flexispira taxa 1, 4 and 5 and the type strain of H. trogontum (ATCC 700114T) had highly related characteristics that differed from those of the reference strains of taxa 2, 3 and 8 and H. bilis. Furthermore, a high DNA–DNA binding rate was found, in dot-blot hybridization studies, between the Finnish porcine strains, taxa 1, 4 and 5 reference strains and H. trogontum ATCC 700114T. In conclusion, polyphasic characterization of novel porcine flexispira isolates and previously described taxa 1, 4 and 5 reference strains showed that they all belong to a validly described species, H. trogontum, and that the taxonomy of known flexispiras is less complicated than proposed on the basis of 16S rDNA sequence analysis.

Sequence analysis of rpoB, the gene encoding the β-subunit of RNA polymerase, was used in a phylogenetic investigation of nine species from the genera Ehrlichia, Neorickettsia, Wolbachia and Anaplasma. The complete nucleotide sequences obtained for Anaplasma phagocytophilum (HGE agent), Ehrlichia chaffeensis, Neorickettsia sennetsu, Neorickettsia risticii, Anaplasma marginale and Wolbachia pipientis were amongst the longest rpoB sequences in GenBank and ranged from 4074 bp for N. sennetsu to 4311 bp for W. pipientis. Additional partial rpoB sequences were obtained for Ehrlichia canis, Ehrlichia ruminantium and Ehrlichia muris. Identical phylogenetic trees were inferred from multiple sequence alignments of the nucleotide sequences and the derived amino acid sequences using either distance, maximum-likelihood or parsimony methods. This study confirms the phylogeny previously inferred from sequence analyses of the 16S rRNA gene, groESL and gltA and allows the confirmation of four monophyletic clades. The rpoB nucleotide sequences were more variable than the 16S rRNA gene and groESL sequences at the species level.

The ascomycete genus Taphrina Fries comprises nearly 100 species recognized by their mycelial states when parasitic on different vascular plants. Whereas the filamentous state is strictly phytoparasitic, the yeast state is saprobic and can be cultured on artificial media. Taphrina species are differentiated mainly on the basis of host range and geographical distribution, type and site of infection and morphology of the sexual stage in infected tissue. However, there has been little progress in the systematics of the genus in recent years, mainly because of the scarcity of molecular studies and available cultures. The main aim of the present study was the reappraisal of species boundaries in Taphrina based on the genetic characterization of cultures (yeast states) that represent about one-third of the currently recognized species. The molecular methods used were (i) PCR fingerprinting using single primers for microsatellite regions and (ii) determination of nucleotide sequences of two approx. 600 bp nuclear rDNA regions, the 5′ end of the 26S rRNA gene (D1/D2 domains) and the internal transcribed spacer region (which includes the 5.8S rRNA gene). Sequencing results confirmed the monophyly of the genus (with the probable exclusion of Taphrina vestergrenii) and the combined analysis of the two methods corroborated, in most cases, separation of species defined on the basis of conventional criteria. However, genetic heterogeneity was found within some species and conspecificity was suggested for strains that have been deemed to represent distinct species. Sequences from the ITS region displayed a higher degree of divergence than those of the D1/D2 region between closely related species, but were relatively conserved within species (>99 % identity) and were thus more useful for the effective differentiation of Taphrina species. The results further allowed other topics to be addressed such as the correlation between the molecular phylogenetic clustering of certain species and the respective host plant family and the significance of molecular methods in the accurate diagnosis of the different diseases caused by Taphrina species.