- Volume 54, Issue 3, 2004

A previously unknown Gram-positive, non-spore-forming, non-lipophilic, catalase-positive, irregular rod-shaped bacterium (M/106/00/5T) was isolated, in mixed culture, from the penis of a Caspian seal (Phoca caspica). The strain was a facultative anaerobe that was able to grow at 22 and 42 °C. Comparative 16S rRNA gene sequencing showed that the organism formed a hitherto unknown subline within the genus Corynebacterium. Sequence divergence values of more than 5 % from other described Corynebacterium species, together with phenotypic differences, showed that the unidentified bacterium represents a previously unrecognized member of this genus. On the basis of phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium isolated from a Caspian seal (strain M/106/00/5T=CCUG 44566T=CIP 107965T) be classified as the type strain of a novel species of the genus Corynebacterium, Corynebacterium caspium sp. nov.

The taxonomic status of Bacillus ehimensis and Bacillus chitinolyticus was examined, based on their 16S rDNA sequences, DNA–DNA hybridization and other taxonomic characteristics. A phylogenetic analysis using 16S rDNA sequences revealed that the two species belong to the genus Paenibacillus. In particular, B. ehimensis KCTC 3748T and B. chitinolyticus KCTC 3791T were found to be phylogenetically closely related to Paenibacillus koreensis YC300T (98·3 % sequence similarity) and Paenibacillus chinjuensis WN9T (95·2 % sequence similarity), respectively. DNA–DNA hybridization values between B. ehimensis KCTC 3748T and P. koreensis YC300T were less than 26 %. An experiment using Paenibacillus-specific PCR primers, PAEN515F and 1377R, revealed that B. ehimensis and B. chitinolyticus had the same amplified 16S rDNA fragment as members of the genus Paenibacillus. Accordingly, it is proposed that B. ehimensis and B. chitinolyticus be transferred to the genus Paenibacillus as Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb. nov., respectively.

Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin. During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found. On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 %. Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 %. DNA–DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation. A mean fatty acid profile could be calculated for each group. Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species. Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests. Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp. nov. (type strain, LMG 21881T=DSM 15866T) and Bacillus gelatini sp. nov. (type strain, LMG 21880T=DSM 15865T).

The previously proposed species Clostridium hastiforme and Tissierella praeacuta appear to be similar from their published descriptions. Accordingly, the aim of the current study was to perform phenotypic and genetic analyses of the type strains of both species, in order to clarify their taxonomic positions. The type strains of C. hastiforme (DSM 5675T) and T. praeacuta (NCTC 11158T) exhibited identical biochemical profiles and their 16S rRNA gene sequences displayed 99·9 % similarity. DNA–DNA hybridization was also estimated to be 96·5 %. Thus, it was concluded that C. hastiforme and T. praeacuta are synonyms, where T. praeacuta has priority. An emended description of the genus Tissierella is also given.

A strict anaerobe (strain GB8-1T) that degraded straight-chain fatty acids with C4–C18 in syntrophic association with methanogens was isolated from an up-flow anaerobic sludge blanket reactor treating beer wastewater. Strain GB8-1T degraded 1 mol butyrate into about 2 mol acetate and 1 mol (presumably) H2 in co-culture with a methanogen. Neither branched-chain fatty acids nor benzoate could be degraded. Strain GB8-1T could grow on crotonate in pure culture and converted 1 mol crotonate to 0·5 mol butyrate and 1 mol acetate. Generation time was about 11 h when grown on crotonate at 37 °C. Fumarate, sulfate, thiosulfate, sulfur and nitrate could not serve as electron acceptors for strain GB8-1T to degrade butyrate. Cells of strain GB8-1T were curved rods with Gram-negative cell walls; no spores were observed. The DNA G+C content was 46·6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GB8-1T was related most closely to the fatty acid-oxidizing, syntrophic bacterium Syntrophomonas sapovorans DSM 3441T; however, the relationship was not very close (95·4 % sequence similarity). Some phenotypic features also differentiated strain GB8-1T from Syntrophomonas sapovorans DSM 3441T. Therefore, a novel species, Syntrophomonas curvata sp. nov., is proposed. The type strain is GB8-1T (=CGMCC 1.5010T=DSM 15682T).

Phylogenetic trees of the anamorphic basidiomycetous yeast genus Trichosporon Behrend, based on molecular sequence analysis of the internal transcribed spacer region and the D1/D2 region of the large subunit of ribosomal (26S) DNA, are presented. This study includes three novel species from soils, Trichosporon vadense sp. nov. (type strain, CBS 8901T), Trichosporon smithiae sp. nov. (type strain, CBS 8370T) and Trichosporon gamsii sp. nov. (type strain, CBS 8245T), one novel species from an insect, Trichosporon scarabaeorum sp. nov. (type strain, CBS 5601T) and one species of unknown origin, Trichosporon dehoogii sp. nov. (type strain, CBS 8686T). The phylogenetic positions and physiological characteristics that distinguish the new taxa from related species, based partly on growth tests that are not traditionally used in yeast taxonomy (uric acid, ethylamine, l-4-hydroxyproline, tyramine and l-phenylalanine as sources of carbon and nitrogen, and polygalacturonate, quinate, 4-ethylphenol, phloroglucinol, 2,3-dihydroxybenzoate and orcinol as sole carbon sources), are discussed. Assimilation of l-rhamnose and erythritol and maximum growth temperature were also used to delineate species.

Seven phylloplane yeast strains that were collected in the Arrábida Natural Park, Portugal, and identified preliminarily as Bullera alba, the anamorphic stage of Bulleromyces albus, were investigated. In contrast to Bulleromyces albus, these isolates produced a brownish pigment when grown on potato dextrose agar. The pigment caused darkening of the cultures and diffused into the culture medium. Mating studies revealed that the Arrábida isolates did not react with the different mating types of Bulleromyces albus, but were sexually compatible with them and produced mycelium with clamp connections, haustoria and transversally septate basidia that ejected the basidiospores. Various taxonomic criteria that were evaluated during the present study and comparison with other sexual taxa of the Tremellales indicated that this teleomorph should be classified in a novel genus. Therefore, Auriculibuller fuscus gen. nov., sp. nov. (type strain, PYCC 5690T=CBS 9648T) is proposed. In addition, during the course of this investigation, a member of a novel Bullera species, Bullera japonica sp. nov. (type strain, PYCC 4534T=CBS 2013T), was found among collection isolates that were identified formerly as Bullera alba. In molecular phylogenetic analysis of the D1/D2 domains of the 26S rDNA and the internal transcribed spacer region, the two taxa were found to be closely related, but distinct at the species level.

Nucleotide sequence analyses of the hymenomycetous yeasts demonstrated that Hyalodendron lignicola should be considered as a member of the genus Trichosporon within the Trichosporonales and that Trichosporon pullulans is associated closely with the Cystofilobasidiales, rather than the Trichosporonales. Accordingly, the following proposals are made: Trichosporon lignicola comb. nov. and Guehomyces gen. nov., to accommodate Guehomyces pullulans comb. nov. in the Cystofilobasidiales.

Analysis of 16S rRNA gene sequences has been the method generally used to study the evolution and phylogeny of bacteria. Phylogenetic analysis of the 16S rRNA gene has shown the position of the genus Helicobacter in the ε-subclass of the Proteobacteria. Because 16S rRNA-based phylogeny does not always correspond to the results of polyphasic taxonomy, and the related species cannot always be separated, new phylogenetic markers for Helicobacter species are needed. In this study, conserved partial (600 bp) 60 kDa heat-shock protein (HSP60) sequences were used to study the phylogeny of 37 strains of gastric and enterohepatic Helicobacter species, including type strains of 15 Helicobacter species with validly published names, reference strains of flexispira taxa and Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis and canine flexispira strains. The partial HSP60 gene sequence proved to be a useful phylogenetic marker for the genus Helicobacter, providing a means of differentiating all 15 Helicobacter species analysed. In the resulting phylogenetic tree, gastric Helicobacter species and enterohepatic species with flexispira morphology formed tight, separate clusters. In general, HSP60 sequence similarities between Helicobacter species were significantly lower than the corresponding 16S rRNA gene sequence similarities, indicating a better resolution for species identification. In addition, a specific PCR method for identifying H. salomonis was developed based on the partial HSP60 sequence.

tRNALeu UAA (trnL) intron sequences are used as genetic markers for differentiating cyanobacteria and for constructing phylogenies, since the introns are thought to be more variable among close relatives than is the 16S rRNA gene, the conventional phylogenetic marker. The evolution of trnL intron sequences and their utility as a phylogenetic marker were analysed among heterocystous cyanobacteria with maximum-parsimony, maximum-likelihood and Bayesian inference by comparing their evolutionary information to that of the 16S rRNA gene. Trees inferred from the 16S rRNA gene and the distribution of two repeat classes in the P6b stem–loop of the trnL intron were in clear conflict. The results show that, while similar heptanucleotide repeat classes I and II in the P6b stem–loop of the trnL intron could be found among distant relatives, some close relatives harboured different repeat classes with a high sequence difference. Moreover, heptanucleotide repeat class II and other sequences from the P6b stem–loop of the trnL intron interrupted several other intergenic regions in the genomes of heterocystous cyanobacteria. Cluster analyses based on conserved intron sequences without loops P6b, P9 and parts of P5 corresponded in most clades to the 16S rRNA gene phylogeny, although the relationships were not resolved well, according to low bootstrap support. Thus, the hypervariable loop sequences of the trnL intron, especially the P6b stem–loop, cannot be used for phylogenetic analysis and conclusions cannot be drawn about species relationships on the basis of these elements. Evolutionary scenarios are discussed considering the origin of the repeats.

In recent years, the planctomycetes have been recognized as a phylum of environmentally important bacteria with habitats ranging from soil and freshwater to marine ecosystems. The planctomycetes form an independent phylum within the bacterial domain, whose exact phylogenetic position remains controversial. With the completion of sequencing of the genome of ‘Rhodopirellula baltica’ SH 1, it is now possible to re-evaluate the phylogeny of the planctomycetes based on multiple genes and genome trees in addition to single genes like the 16S rRNA or the elongation factor Tu. Here, evidence is presented based on the concatenated amino acid sequences of ribosomal proteins and DNA-directed RNA polymerase subunits from ‘Rhodopirellula baltica’ SH 1 and more than 90 other publicly available genomes that support a relationship of the Planctomycetes and the Chlamydiae. Affiliation of ‘Rhodopirellula baltica’ SH 1 and the Chlamydiae was reasonably stable regarding site selection since, during stepwise filtering of less-conserved sites from the alignments, it was only broken when rigorous filtering was applied. In a few cases, ‘Rhodopirellula baltica’ SH 1 shifted to a deep branching position adjacent to the Thermotoga/Aquifex clade. These findings are in agreement with recent publications, but the deep branching position was dependent on site selection and treeing algorithm and thus not stable. A genome tree calculated from normalized blastp scores did not confirm a close relationship of ‘Rhodopirellula baltica’ SH 1 and the Chlamydiae, but also indicated that the Planctomycetes do not emerge at the very root of the Bacteria. Therefore, these analyses rather contradict a deep branching position of the Planctomycetes within the bacterial domain and reaffirm their earlier proposed relatedness to the Chlamydiae.

The phylogenetic position of the Mollicutes has been re-examined by using phosphoglycerate kinase (Pgk) amino acid sequences. Hitherto unpublished sequences from Mycoplasma mycoides subsp. mycoides, Mycoplasma hyopneumoniae and Spiroplasma citri were included in the analysis. Phylogenetic trees based on Pgk data indicated a monophyletic origin for the Mollicutes within the Firmicutes, whereas Bacilli (Firmicutes) and Clostridia (Firmicutes) appeared to be paraphyletic. With two exceptions, i.e. Thermotoga (Thermotogae) and Fusobacterium (Fusobacteria), which clustered within the Firmicutes, comparative analyses show that at a low taxonomic level, the resolved phylogenetic relationships that were inferred from both the Pgk protein and 16S rRNA gene sequence data are congruent.

The genus Spiroplasma (helical mollicutes: Bacteria: Firmicutes: Mollicutes: Entomoplasmatales: Spiroplasmataceae) is associated primarily with insects. The Mycoplasma mycoides cluster (sensu Weisburg et al. 1989 and Johansson and Pettersson 2002 ) is a group of mollicutes that includes the type species – Mycoplasma mycoides – of Mycoplasmatales, Mycoplasmataceae and Mycoplasma. This cluster, associated solely with ruminants, contains five other species and subspecies. Earlier phylogenetic reconstructions based on partial 16S rDNA sequences and a limited sample of Spiroplasma and Mycoplasma sequences suggested that the genus Mycoplasma was polyphyletic, as the M. mycoides cluster and the grouping that consisted of the hominis and pneumoniae groups of Mycoplasma species were widely separated phylogenetically and the M. mycoides cluster was allied with Spiroplasma. It is shown here that the M. mycoides cluster arose from Spiroplasma through an intermediate group of non-helical spiroplasmal descendants – the Entomoplasmataceae. As this conclusion has profound implications in the taxonomy of Mollicutes, a detailed phylogenetic study of Spiroplasma and its non-helical descendants was undertaken. These analyses, done with maximum-parsimony, provide cladistic status; a new nomenclature is introduced here, based on ‘bottom-up’ rather than ‘top-down’ clade classification. The order Entomoplasmatales consists of four major clades: (i) the Mycoides–Entomoplasmataceae clade, which contains M. mycoides and its allies and Entomoplasma and Mesoplasma species and is a sister lineage to (ii) the Apis clade of Spiroplasma. Spiroplasma and the Entomoplasmataceae are paraphyletic, but this status does not diminish their phylogenetic usefulness. Five species that were previously unclassified phylogenetically are basal to the Apis clade sensu strictu and to the Mycoides clade. One of these species, Spiroplasma sp. TIUS-1, has very poor helicity and a very small genome (840 kbp); this putative species can be envisioned as a ‘missing link’ in the evolution of the Mycoides–Entomoplasmataceae clade. The other two Spiroplasma clades are: (iii) the Citri–Chrysopicola–Mirum clade (serogroups I, II, V and VIII) and (iv) the ixodetis clade (serogroup VI). As Mesoplasma lactucae represents a basal divergence within the Mycoides–Entomoplasmataceae clade, and as Entomoplasma freundtii is basal to the Mycoides clade, M. mycoides and its allies must have arisen from an ancestor in the Entomoplasmataceae. The paraphyletic grouping that consists of the Hominis and Pneumoniae groups (sensu Johansson & Pettersson 2002 ) of Mycoplasma species contains the ancestral roots of Ureaplasma spp. and haemoplasmas. This clade is a sister lineage to the Entomoplasmatales clade. Serological classifications of spiroplasma are very highly supported by the trees presented. Genome size and G+C content of micro-organismal DNA were moderately conserved, but there have been frequent and polyphyletically distributed genome reductions. Sterol requirements were polyphyletic, as was the ability to grow in the presence of polyoxyethylene sorbitan-supplemented, but not serum-supplemented, media. As this character is not phylogenetically distributed, Mesoplasma and Entomoplasma should be combined into a single genus. The phylogenetic trees presented here confirm previous reports of polyphyly of the genus Mycoplasma. As both clades of Mycoplasma contain several species of great practical importance, a change of the genus name for species in either clade would have immense practical implications. In addition, a change of the genus name for M. mycoides would have to be approved by the Judicial Commission. For these reasons, the Linnaean and phylogenetic classifications of Mycoplasma must for now be discrepant.

This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0·58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.

The bacterial genus Microsphaera Yoshimi et al. 1996 is illegitimate because of priority of the fungal genus Microsphaera (Wallr.) Lév. [Principle 2, Rule 51b(4) of the Bacteriological Code]. Therefore, a new genus name, Nakamurella, is proposed for the bacterial genus. The type species Microsphaera multipartita Yoshimi et al. 1996 becomes Nakamurella multipartita gen. nov., comb. nov. Due to the illegitimacy of the only genus in the family Microsphaeraceae Rainey et al. 1997, this family name is replaced by the new bacterial family name Nakamurellaceae.