- Volume 53, Issue 3, 2003

Three clusters of isolates have previously been defined within the species Comamonas terrigena, on the basis of DNA–rRNA and DNA–DNA hybridization data, and of protein electrophoretic patterns and immunotyping. More detailed characterization in the current study shows that representatives of these three groups can also be differentiated phenotypically from each other. Strains of C. terrigena sensu stricto (C. terrigena DNA group 1) are pyrrolidone aminopeptidase-positive, do not grow at 40 °C, are l-alanine-positive and are always negative for 4-hydroxybenzoate. Strains of C. terrigena DNA groups 2 and 3 are pyrrolidone aminopeptidase-negative; the former is the only group that is tyrosine-negative, and only the latter can grow at 42 °C (with an optimal growth temperature of 40 °C). These findings are corroborated by differences in 16S rDNA sequence and tRNA intergenic spacer lengths. Therefore, it is proposed to rename C. terrigena DNA group 2 [containing former Aquaspirillum aquaticum and E. Falsen (EF) group 10 strains] as Comamonas aquatica sp. nov., and C. terrigena DNA group 3 (containing former EF group 10 strains) as Comamonas kerstersii sp. nov.

A group of seven sucrose-negative Aeromonas strains (referred to as group Au) isolated from the internal organs of septicaemic farmed frogs (Rana rugulosa) in Thailand was subjected to a polyphasic taxonomic study including fluorescent amplified fragment length polymorphism (FAFLP) and ERIC-PCR fingerprinting, 16S rDNA sequencing, microplate DNA–DNA hybridizations and extensive phenotypic characterization. Comparison of FAFLP and ERIC-PCR fingerprints indicated that the group Au isolates belonged to the species Aeromonas hydrophila DNA hybridization group (HG) 1 in which they represent a genotypic subgroup closely affiliated to A. hydrophila subsp. hydrophila and subsp. dhakensis. One representative of the Au group exhibited ⩾99·0 % 16S rDNA sequence similarity with the type strains of the two A. hydrophila subspecies. DNA–DNA hybridization with type and reference strains of all known Aeromonas taxa revealed that the Au group represented a homogeneous taxon that exhibited the highest relatedness with members of the two A. hydrophila subspecies, ranging from 75 to 93 %. Phenotypic characterization on the basis of 152 features further revealed that the Au group isolates differed from A. hydrophila subsp. hydrophila or subsp. dhakensis in a total of 13 biochemical properties. Of these, assimilation of l-glycine and isobutyrate as sole carbon source, acid production from salicin and d-sucrose, and aesculin hydrolysis were of diagnostic value. From the results of this study, it can be concluded that the Aeromonas frog isolates of the Au group represent a new subspecies of A. hydrophila, for which the name Aeromonas hydrophila subsp. ranae subsp. nov. is proposed. Its type strain is Au-1D12T (=LMG 19707T=CCUG 46211T).

A format for the description of single novel species is proposed, which should facilitate the reviewing process by assisting the provision of data in a standardized form. The abstract must be short and concise, highlighting phylogenetic position, morphology and chemotaxonomy for genus affiliation, the genotypic and phenotypic basis for species differentiation, and the name and deposition numbers from two public culture collections in different countries for the type strain: A Gram-negative, rod-shaped, non-spore-forming bacterium (Iso 196T) was isolated from chicken faeces. On the basis of 16S rRNA gene sequence similarity, strain Iso 196T was shown to belong to the α-2 subclass of the Proteobacteria related to Ochrobactrum tritici (95·6 %), Ochrobactrum grignonense (95·0 %) and Ochrobactrum anthropi (94·6 %), and the phylogenetic distance from any validly described species within the genus Brucella was less than 95 %. Chemotaxonomic data (major ubiquinone – Q-10; major polyamines – spermidine and putrescine; major polar lipids – phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine; major fatty acids – C18 : 1 ω7c and C19 : 0 cyclo ω8c) supported the affiliation of strain Iso 196T to the genus Ochrobactrum. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Iso 196T from the four validly published Ochrobactrum species. Iso 196T therefore represents a new species, for which the name Ochrobactrum gallinifaecis sp. nov. is proposed, with the type strain Iso 196T (=DSM 15295T =CIP 107753T).

‘Streptococcus bovis/Streptococcus equinus’ is a large bacterial complex including different species frequently isolated from infections of humans (Streptococcus gallolyticus, Streptococcus infantarius) or animals (S. bovis, S. equinus, Streptococcus alactolyticus). The separation of S. bovis into three different biotypes has been partially correlated with genetic differentiation. In addition, recent advances in bacterial phylogeny have led to the inclusion of Streptococcus macedonicus and Streptococcus waius in this complex. The aim of this study was to improve physiological differentiation between species related to the complex and to clarify their respective phylogenetic positions. In this study, physiological, genetic and phylogenetic analyses of a set of 88 streptococcal strains were performed. The diversity of strains of S. bovis biotype II was analysed, and it was confirmed that they belong to different species, either S. equinus or S. infantarius. It was demonstrated that S. gallolyticus, S. bovis biotype II.2, S. macedonicus and S. waius form a single DNA cluster separated into three different subspecies. They are delineated by different biochemical traits, limited DNA–DNA relatedness and noticeable divergence in 16S rDNA sequences. According to the current definition of species, the names S. gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. pasteurianus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov. are proposed for these three subspecies.

Phenotypic and genotypic characterizations of Lactobacillus maltaromicus strains DSM 20342T and DSM 20344 provided evidence for the reclassification of this species in the genus Carnobacterium. Moreover, phenotypic and genotypic comparisons made between L. maltaromicus and Carnobacterium piscicola highlighted that these two species should be considered synonyms. For these reasons, the species Carnobacterium maltaromaticum comb. nov. (type strain DSM 20342T=ATCC 27865T=CCUG 30142T=CIP 103135T=JCM 1154T=LMG 6903T=NRRL B-14852T) is proposed to accommodate L. maltaromicus and C. piscicola.

A hitherto undescribed Actinomyces-like bacterium was isolated from human urine. Based on its biochemical characteristics, the unidentified bacterium did not correspond to any currently described Actinomyces species or related taxa. Comparative 16S rRNA gene sequencing showed that the unknown bacterium exhibits a specific phylogenetic association with the genus Actinobaculum, but a sequence divergence of >5 % from the two currently recognized members of this genus, Actinobaculum schaalii and Actinobaculum suis, demonstrates that it represents a distinct species. Based on both phenotypic and 16S rRNA gene sequence considerations, it is proposed that the unknown bacterium from urine should be classified as a novel species, Actinobaculum urinale sp. nov. The type strain of Actinobaculum urinale is CCUG 46093T (=CIP 107424T).

A facultatively anaerobic, Gram-positive, coccoid, non-spore-forming, non-motile, catalase-negative bacterium was isolated from the uropygial (preen) gland of wild Red-billed Woodhoopoes (Phoeniculus purpureus) and designated strain JLB-1T. Physiological and biochemical testing suggested that this homofermentative, lactic-acid-producing bacterium could belong to the genus Enterococcus or the genus Streptococcus. Comparison of the 16S rRNA gene sequence of strain JLB-1T with other 16S rDNA sequences in the GenBank database by blast analysis showed that its closest relatives are Enterococcus faecium, Enterococcus avium and Enterococcus asini. Strain JLB-1T may be differentiated from these species by the fact that it does not produce acid from lactose, d-mannitol, d(+)-melezitose or d-sorbitol. Furthermore, it does not hydrolyse arginine or hippurate and cannot grow in the presence of 6·5 % NaCl or 40 % bile. It differs from the streptococci in that it does not lyse erythrocytes. Strain JLB-1T is a novel member of the enterococci, for which the name Enterococcus phoeniculicola sp. nov. is proposed. The type strain is JLB-1T (=ATCC BAA-412T=DSM 14726T).

A Gram-positive, rod-shaped, endospore-forming, halophilic bacterium (strain HSL-3T) was isolated from a salt lake near Hwajinpo beach on the East Sea in Korea and was subjected to a polyphasic taxonomic study. Strain HSL-3T grew optimally in the presence of 2–10 % (w/v) NaCl. Strain HSL-3T showed poor growth in the absence of NaCl and grew in the presence of less than 23 % NaCl. The cell wall peptidoglycan type of strain HSL-3T was A4β based on l-orn–d-Asp. The predominant menaquinone found in strain HSL-3T was menaquinone-7 (MK-7). Strain HSL-3T had a cellular fatty acid profile containing large amounts of branched fatty acids; the major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0. The DNA G+C content of strain HSL-3T was 45 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain HSL-3T falls within the radiation of the cluster comprising Halobacillus species. Strain HSL-3T exhibited levels of 16S rDNA similarity of 97·4–98·4 % to the type strains of Halobacillus species. Levels of DNA–DNA relatedness between strain HSL-3T and the type strains of all validly named Halobacillus species were in the range 7·3–9·2 %. On the basis of phenotypic and phylogenetic data and the genomic distinctiveness, strain HSL-3T (=KCCM 41590T=JCM 11546T) should be placed in the genus Halobacillus as the type strain of a novel species, for which the name Halobacillus salinus sp. nov. is proposed.

A bacterial strain, strain IMMIB R-5091T, isolated from a cosmetic dye was characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing showed that the isolate constitutes a distinct subline within the genus Corynebacterium, displaying >2·6 % sequence divergence from established species. The isolate could be distinguished from other members of the genus Corynebacterium by biochemical tests. Based on both phenotypic and phylogenetic evidence, it is proposed that strain IMMIB R-5091T (=DSM 44530T=NRRL B-24142T) be classified as the type strain of a novel species, Corynebacterium glaucum sp. nov.

A novel marine lactic acid rod bacterium has been described for eight strains isolated from living and decomposing marine organisms collected from temperate and subtropical areas of Japan. The isolates were Gram-positive, catalase-negative, non-sporulating and motile with peritrichous flagella. They were slightly halophilic, highly halotolerant and alkaliphilic; the optimum NaCl concentration for growth was 2·0–3·75 % (w/v) with a range from 0 to 17·0–20·5 % (depending on the strain); the optimum pH was between 8·0 and 9·5 with a range from 6·0 to 10·0. They were psychrotolerant, growing well at −1·8 °C with a maximum at 40–45 °C and the optimum at 37–40 °C. Lactate yields were 87–100 % per consumed glucose; the residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The product composition was markedly affected by the pH of fermentation medium; at higher pH, the yield of lactate decreased (60–65 % at pH 9·0) and that of other products increased conversely. The cell-wall peptidoglycan type was type A4β, Orn-d-Glu, whereas that of the genus Alkalibacterium, the phylogenetically closest lactic acid bacterium, was type A4β, Orn-d-Asp. The major cellular fatty acids were C16 : 0, C16 : 1Δ9, C18 : 0 and C18 : 1Δ9 (oleic acid). The G+C content of the DNA was 34·6–36·2 mol%. The eight isolates were phenotypically homogeneous and formed a single genomic species. The 16S rRNA gene sequence analysis indicated that the isolates constituted an independent phylogenetic lineage within the radiation of lactic acid bacteria with 96·2 % similarity to the genus Alkalibacterium. The secondary structure and the nucleotide sequence of the V6 region of the 16S rRNA were characteristic of the organism among other related lactic acid genera. On the bases of phenotypic and phylogenetic distinctness, the organism was proposed to belong to a new genus and species, Marinilactibacillus psychrotolerans gen. nov., sp. nov. The type strain, M13-2T (G+C=36·2 mol%), has been deposited in the IAM, NBRC, NCIMB and NRIC culture collections as IAM 14980T, NBRC 100002T, NCIMB 13873T and NRIC 0510T, respectively.

Strain HAG 010767T was isolated from desert soil from Namibia during a screening programme. On the basis of analysis of 16S rDNA, the principal amino acid of the peptidoglycan, cell-wall sugars, fatty acids and polar lipids, it was possible to identify this strain as a member of the genus Actinomadura. Although DNA–DNA reassociation experiments revealed 72 % DNA similarity between strain HAG 010767T and Actinomadura kijaniata DSM 43764T, significant differences in the colour of the mycelium and physiological properties indicate that strain HAG 010767T represents a novel species of this genus, for which the name Actinomadura namibiensis sp. nov. is proposed. The type strain is strain HAG 010767T (=DSM 44197T=NRRL B-24153T).

In a project concerning bacterial colonization of experimental wall paintings, a large number of isolates have been acquired with high similarities in their whole-cell protein patterns obtained after SDS-PAGE. Of this group, four strains, designated V2-BIII-A2T, V2-BI-A9, V2-BI-04 and V2-BII-A8, were chosen for further characterization. Banding patterns obtained after ERIC-PCR were barely distinguishable among these four strains, indicating their affiliation within a single species. The isolates also displayed nearly identical biochemical and physiological features. The chemotaxonomic characteristics, including polar lipids, quinone systems, cell-wall diamino acid composition and fatty acid profiles, were in good agreement with those of numerous previously described Bacillus species. 16S rDNA analysis of strain V2-BIII-A2T showed that this bacterium belongs to the genus Bacillus, with highest sequence similarities to Bacillus megaterium (94·6 %), Bacillus flexus (94·4 %) and the alkaliphilic Bacillus cohnii (94·2 %). Based on almost identical biochemical, physiological and chemotaxonomic traits, ERIC-PCR-generated genomic fingerprints and comparative 16S rDNA sequence analysis, it is demonstrated that the four isolates represent a novel species of the genus Bacillus, for which the name Bacillus barbaricus sp. nov. is proposed. The type strain is V2-BIII-A2T (=CCM 4982T=DSM 14730T).

An actinomycete with blue substrate mycelium was isolated from a soil sample in Cape Town, South Africa, and designated strain PK-BlueT. The colour of the substrate mycelium was not sensitive to changes in pH. The organism produced hairy spores in Spirales-type spore chains. Chemical taxonomy indicated that it belonged to the genus Streptomyces. Strain PK-BlueT produced no diffusible pigments other than melanin, grew at 45 °C, did not degrade adenine and exhibited no antibacterial activity against Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa. Analysis of its 16S rRNA gene sequence and the results of physiological tests showed that strain PK-BlueT (=DSM 41797T=ATCC BAA-411T) represents the type strain of a novel species of Streptomyces, for which the name Streptomyces speibonae sp. nov. is proposed.

An unknown Gram-positive, catalase-negative, facultatively anaerobic, non-spore-forming, rod-shaped bacterium originating from semen of a pig was characterized using phenotypic, molecular chemical and molecular phylogenetic methods. Chemical studies revealed the presence of a directly cross-linked cell wall murein based on l-lysine and a DNA G+C content of 39 mol%. Comparative 16S rRNA gene sequencing showed that the unidentified rod-shaped organism formed a hitherto unknown subline related, albeit loosely, to Alkalibacterium olivapovliticus, Alloiococcus otitis, Dolosigranulum pigrum and related organisms, in the low-G+C-content Gram-positive bacteria. However, sequence divergence values of >11 % from these recognized taxa clearly indicated that the novel bacterium represents a separate genus. Based on phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium from pig semen be classified as a new genus and species, Allofustis seminis gen. nov., sp. nov. The type strain is strain 01-570-1T (=CCUG 45438T=CIP 107425T).

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches’-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches’-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called ‘almond brooming’ in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name ‘Candidatus Phytoplasma phoenicium’ is proposed for the phytoplasma associated with almond witches’-broom in Lebanon and Iran.

A keratinolytic, nocardioform actinomycete, strain D2T, was isolated from Kuwait marsh soil by the bait technique using animal wool. Strain D2T was an aerobic, Gram-positive organism that produced light-grey aerial mycelium but no specific spore chains. 16S rDNA sequence analyses and chemotaxonomic markers were consistent with the classification of strain D2T in the genus Amycolatopsis, i.e. it had meso-diaminopimelic acid in its peptidoglycan, arabinose and galactose as its diagnostic sugars, the polar lipids phosphatidylinositol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine and diphosphatidylglycerol, menaquinone MK-9(H4) and an iso-/anteiso-branched fatty acid pattern combined with 10-methyl-branched and 2-hydroxy-branched fatty acids. Amycolatopsis japonica was the closest phylogenetic neighbour of strain D2T, showing 99·4 % 16S rDNA sequence similarity with the novel strain. A. japonica and strain D2T could be clearly separated from each other on the basis of their low DNA–DNA reassociation value (55·9 %). These data, together with its distinct physiological traits, led to the conclusion that strain D2T represented a novel species within the genus Amycolatopsis, for which the name Amycolatopsis keratiniphila (type strain D2T=DSM 44409T=NRRL B24117T) is proposed.

Three novel species of the genus Rhodotorula are described. Rhodotorula benthica sp. nov. (type strain JCM 10901T =SY-91T) and Rhodotorula calyptogenae sp. nov. (type strain JCM 10899T =SY-86T) were respectively isolated from the tubeworm Lamellibrachia sp. and the giant white clam Calyptogena sp., collected from the deep-sea floor of the Pacific Ocean off Japan. Rhodotorula lysiniphila sp. nov. (type strain JCM 5951T) is proposed for strains isolated previously in Japan and Pakistan. The three species were placed phylogenetically into a species complex comprising Rhodotorula laryngis, Rhodotorula minuta, Rhodotorula pallida and Rhodotorula slooffiae. R. minuta and R. slooffiae are closely related in both the D1/D2 region of the 26S rDNA and the internal transcribed spacer and 5·8S rDNA regions. R. benthica and R. laryngis were closer to R. pallida based on the D1/D2 region. Other relationships were not clear.

Three mycocinogenic strains representing the genus Cryptococcus were isolated on glucuronate agar from plants and turf collected in the Prioksko-terrasny biosphere reserve (Russia). These isolates fit the standard description of Cryptococcus laurentii, but differ from its type strain in both their mycocin-sensitivity profiles and the killing patterns of their mycocins. Sequence analyses of the D1/D2 domain of the 26S rDNA and of the internal transcribed spacer region confirmed that these isolates represent two novel species, for which the names Cryptococcus nemorosus sp. nov. (type strain VKM Y-2906T) and Cryptococcus perniciosus sp. nov. (type strain VKM Y-2905T) are proposed. Morphological, physiological and biochemical characteristics, as well as mycocinotyping and molecular analysis, show a close affinity between these two novel anamorphic species and the teleomorphic species Papiliotrema bandonii (Tremellales).