- Volume 55, Issue 1, 2005

Two strains of an unidentified, Gram-positive, catalase-negative, chain-forming, coccus-shaped organism recovered from seals were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria the strains were tentatively identified as streptococci but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies showed that the strains were closely related to each other and confirmed their placement in the genus Streptococcus. Sequence divergence values of >5 % with reference streptococcal species demonstrated the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed that the two organisms were closely related to each other but were different from all currently defined streptococcal species. Based on biochemical criteria, molecular chemical and molecular genetic evidence, it is proposed that the unknown isolates from seals be assigned to a novel species of the genus Streptococcus, Streptococcus marimammalium sp. nov. The type strain is M54/01/1T (=CCUG 48494T=CIP 108309T).

Pinus silvestris and Pinus halepensis trees grown in Germany and Spain, respectively, showing abnormal shoot branching, dwarfed needles and other symptoms were examined for the presence of plant-pathogenic mollicutes (phytoplasmas). While phytoplasmas could not be detected unambiguously with microscopical methods, PCR amplification using universal phytoplasma primers yielded positive results. Samples collected from symptomatic and non-symptomatic plant parts of both symptomatic Pinus silvestris and Pinus halepensis trees tested positive. Also, surrounding non-symptomatic trees proved to be phytoplasma-infected. Comparisons revealed that the 16S rRNA gene sequences of the phytoplasmas identified in Pinus silvestris and Pinus halepensis were nearly identical. However, the pine phytoplasma is only distantly related to other phytoplasmas. The closest relatives are members of the palm lethal yellowing and rice yellow dwarf groups and ‘Candidatus Phytoplasma castaneae’, which share between 94·5 and 96·6 % 16S rRNA gene sequence similarity. From these data it can be concluded that the phytoplasmas identified in the two Pinus species represent a coherent but discrete taxon; it is proposed that this taxon be distinguished at putative species level under the name ‘Candidatus Phytoplasma pini’.

Eight strains of aerobic, spore-forming, Gram-positive, moderately halophilic bacteria were isolated from sauce (nam-pla and bu-du) produced in Thailand by the fermentation of fish. They grew optimally in the presence of 10 % NaCl, at 37 °C and pH 7·0. A diagnostic diamino acid, meso-diaminopimelic acid, was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7. The major cellular fatty acids were anteiso-C15 : 0 and iso-C16 : 0. Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid were found to be the major polar lipid components. The DNA G+C content was 42–43 mol%. These bacteria were further divided into two groups based on phenotypic characteristics and DNA–DNA similarities. Three strains of Group I were highly affiliated to the type strain of Lentibacillus salicampi in terms of phenotypic characterization and DNA–DNA similarities (96–102 %); accordingly, they were identified as strains of L. salicampi. A representative strain of Group II, strain IS40-3T, was most closely related to L. salicampi in terms of 16S rRNA-based phylogenetic analysis, although five strains of Group II could be distinguished from L. salicampi by means of several phenotypic properties, low 16S rRNA gene sequence similarity (95·2 %) and low DNA–DNA similarities (12–32 %). Therefore, the Group II strains should be included in a novel species of the genus Lentibacillus, for which the name Lentibacillus juripiscarius sp. nov. is proposed. The type strain is IS40-3T (=JCM 12147T=PCU 229T=TISTR 1535T).

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0–120 g l−1, with the optimum at 10–20 g l−1. The temperature range for growth at pH 7·0 was 4–50 °C, with the optimum at 37–40 °C. The optimum pH for growth was 7·0–8·0. The optimum pressure for growth was 0·1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2T (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA–DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

Phylogenetic analysis based on 16S rRNA gene sequences revealed that Thermoactinomyces species with validly published names can be assigned to four clusters or lineages. The type strains of Thermoactinomyces sacchari and Thermoactinomyces putidus were differentiated from the type strains of Thermoactinomyces vulgaris and Thermoactinomyces intermedius by the predominant menaquinone and fatty acid profiles. The type strains of Thermoactinomyces dichotomicus and Thermoactinomyces peptonophilus formed lines of descent distinct from other Thermoactinomyces species. Thermoactinomyces dichotomicus KCTC 3667T was distinguishable from the type strains of Thermoactinomyces vulgaris and Thermoactinomyces intermedius by the contents of two fatty acids, iso-C16 : 0 and iso-C17 : 0. Thermoactinomyces dichotomicus could be distinguished from other Thermoactinomyces species by DNA G+C content and some phenotypic properties, particularly its property of forming a yellow colour. The type strain of Thermoactinomyces peptonophilus was distinguishable from other Thermoactinomyces species by differences in menaquinone profile, major fatty acids, DNA G+C content and some physiological properties including optimal growth temperature. On the basis of these data, the creation of three new genera, Laceyella, Thermoflavimicrobium and Seinonella, is proposed in addition to the genus Thermoactinomyces sensu stricto. The genus Laceyella gen. nov. is proposed to accommodate Thermoactinomyces sacchari and Thermoactinomyces putidus as Laceyella sacchari comb. nov. and Laceyella putida comb. nov., the genus Thermoflavimicrobium gen. nov. is proposed for Thermoactinomyces dichotomicus as Thermoflavimicrobium dichotomicum comb. nov. and the genus Seinonella gen. nov. is proposed for Thermoactinomyces peptonophilus as Seinonella peptonophila comb. nov.

Four strains isolated from Indian dairy products and initially identified as Lactobacillus delbrueckii could not be assigned to a definite subspecies because molecular identification and phenotypic traits did not agree with those of recognized subspecies of L. delbrueckii. Hybridization of total DNA (78–86 % against type strains of the other three subspecies), AFLP and RAPD-PCR fingerprints, phylogenetic analysis based on 16S rRNA gene sequences and sequence analysis of two coding genes (recA and hsp60), together with phenotypic profiles, indicated that the four strains form a coherent cluster and represent a novel subspecies, for which the name Lactobacillus delbrueckii subsp. indicus subsp. nov. is proposed. The type strain is NCC725T (=LMG 22083T=DSM 15996T).

During a search for xylan-degrading micro-organisms, a sporulating bacterium was recovered from xylan-containing agar plates exposed to air in a research laboratory (Salamanca University, Spain). The airborne isolate (designated strain XIL14T) was identified by 16S rRNA gene sequencing as representing a Paenibacillus species most closely related to Paenibacillus illinoisensis JCM 9907T (99·3 % sequence similarity) and Paenibacillus pabuli DSM 3036T (98 % sequence similarity). Phenotypic, chemotaxonomic and DNA–DNA hybridization data indicated that the isolate belongs to a novel species of the genus Paenibacillus. Cells of strain XIL14T were motile, sporulating, rod-shaped, Gram-positive and facultatively anaerobic. The predominant cellular fatty acids were anteiso-C15 : 0 and C16 : 0. The DNA G+C content of strain XIL14T was 50·5 mol%. Growth was observed with many carbohydrates, including xylan, as the only carbon source and gas production was not observed from glucose. Catalase was positive and oxidase was negative. The airborne isolate produced a variety of hydrolytic enzymes, including xylanases, amylases, gelatinase and β-galactosidase. DNA–DNA hybridization levels between strain XIL14T and P. illinoisensis DSM 11733T and P. pabuli DSM 3036T were 43·3 and 36·3 %, respectively. According to the data obtained, strain XIL14T is considered to represent a novel species for which the name Paenibacillus xylanilyticus sp. nov. is proposed (=LMG 21957T=CECT 5839T).

Two actinomycete strains, LK2-10T and LK2-5, which produced single, non-motile spores, were isolated from peat swamp forest soil in Yala Province, Thailand. A polyphasic study was carried out to establish the taxonomic position of these strains. Morphological and chemotaxonomic characteristics of these strains coincided with those of the genus Micromonospora. Phylogenetic analysis using 16S rRNA gene sequences also indicated that these strains should be classified in the genus Micromonospora and clearly separated from their closest relative, Micromonospora nigra DSM 43818T. Furthermore, a combination of DNA–DNA hybridization results and physiological and biochemical properties indicated that these strains were distinguished from all recognized Micromonospora species. These strains therefore represent a novel species, for which the name Micromonospora eburnea sp. nov. is proposed. The type strain is LK2-10T (=JCM 12345T=PCU 238T=DSM 44814T=TISTR 1531T).

A previously undescribed filamentous, beaded, Gram-positive, rod-shaped bacterium was isolated from pus of a human dental abscess. Based on its cellular morphology and the results of biochemical testing the organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species of this genus. Comparative 16S rRNA gene sequencing studies showed the bacterium represents a distinct subline within the genus Actinomyces, clustering within a group of species that includes Actinomyces bovis, the type species of the genus. Sequence divergence values of >8 % with other recognized species within this phylogenetic group clearly demonstrated that the organism represents a hitherto unknown species. Based on biochemical and molecular phylogenetic evidence, it is proposed that the unidentified organism recovered from a dental abscess be classified as a novel species, Actinomyces dentalis sp. nov. The type strain is R18165T (=CCUG 48064T=CIP 108337T).

Chemotaxonomic and morphological characterization of two actinomycete strains, MS1-3T and AS4-2, respectively isolated from moat sediment and scumming activated sludge, was carried out. This characterization clearly demonstrated that strains MS1-3T and AS4-2 belong to the genus Nocardia. 16S rRNA gene sequencing studies showed that these isolates are most closely related to Nocardia beijingensis (98·1–98·3 % similarity), Nocardia brasiliensis (97·9–98·0 %) and Nocardia tenerifensis (97·8–97·9 %). However, the results of DNA–DNA hybridizations and physiological and biochemical tests showed that strains MS1-3T and AS4-2 could be differentiated from their closest phylogenetic relatives both genotypically and phenotypically. It is proposed that the two isolates be classified as representatives of a novel species of Nocardia, Nocardia takedensis sp. nov. The type strain is MS1-3T (=NBRC 100417T=DSM 44801T); AS4-2 (=NBRC 100418=DSM 44802) is a reference strain.

A Gram-positive, rod-shaped, spore-forming bacterium (PAT 05T) was isolated from the rhizosphere of the perennial shrub Atriplex lampa in north-eastern Patagonia, Argentina. Its overall biochemical and physiological characteristics indicated that this strain should be placed in the alkaliphilic Bacillus group. Strain PAT 05T grew at pH 7–10 (optimum pH 8), but not at pH 6. Its DNA G+C content was 39·7 mol%. Sequence analysis of the 16S rRNA gene of PAT 05T revealed the closest match (99·6 % similarity) with Bacillus sp. DSM 8714. The highest level of DNA–DNA relatedness (88·6 %) was also found with this strain. On the basis of 16S rRNA gene sequence similarity and phylogenetic analysis, G+C content and DNA–DNA hybridization data, strain PAT 05T is related at the species level to Bacillus sp. DSM 8714, a member of a group referred as phenon 4a by Nielsen et al. [Nielsen, P., Fritze, D. & Priest, F. G. (1995). Microbiology 141, 1745–1761] , which still lacks taxonomic standing. These results support the proposal of strain PAT 05T (=DSM 16117T=ATCC BAA-965T) as the type strain of Bacillus patagoniensis sp. nov.

Fourteen isolates of two different bacterial species isolated from the surface of smear-ripened cheeses were found to exhibit many characteristics of the genus Arthrobacter. The isolates were aerobic, Gram-positive, catalase-positive, non-spore-forming and non-motile. The cell-wall peptidoglycan contained lysine, alanine and glutamic acid. rrs sequence analysis indicated that the new isolates Re117T and Ca106T are closely related to the Arthrobacter nicotianae group and showed highest sequence similarity (>98 %) to Arthrobacter nicotianae and Arthrobacter protophormiae. However, DNA–DNA hybridization studies indicated that the strains represented two novel genomic species within the genus Arthrobacter and did not belong to A. nicotianae or A. protophormiae (<43 % DNA–DNA relatedness). On the basis of the phylogenetic and phenotypic distinctiveness of the new isolates, these bacteria should be classified as two novel Arthrobacter species, for which the names Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov. are proposed. Type strains have been deposited in culture collections as Arthrobacter bergerei Ca106T (=CIP 108036T=DSM 16367T) and Arthrobacter arilaitensis Re117T (=CIP 108037T=DSM 16368T).

Two novel actinobacteria isolates, designated YIM 70009T and YIM 70081T, were characterized in order to determine their taxonomic position. Cells of strains YIM 70009T and YIM 70081T were cocci, although only the latter were motile. The G+C contents of their DNAs were 64·0 and 64·5 mol%, respectively. On the basis of chemotaxonomic characteristics and 16S rRNA gene sequence analysis, the two isolates were classified in the genus Nesterenkonia. DNA–DNA hybridization and comparison of phenotypic characteristics revealed that strains YIM 70009T and YIM 70081T differed from each other and from known species. Therefore, it is proposed that they represent two separate novel species of the genus Nesterenkonia: Nesterenkonia sandarakina sp. nov. (type strain, YIM 70009T=CCTCC AA 203007T=DSM 15664T=KCTC 19011T) and Nesterenkonia lutea sp. nov. (type strain, YIM 70081T=CCTCC AA 203010T=DSM 15666T=KCTC 19013T).

A novel, psychrotolerant, facultative anaerobe, strain FTR1T, was isolated from Pleistocene ice from the permafrost tunnel in Fox, Alaska. Gram-positive, motile, rod-shaped cells were observed with sizes 0·6–0·7×0·9–1·5 μm. Growth occurred within the pH range 6·5–9·5 with optimum growth at pH 7·3–7·5. The temperature range for growth of the novel isolate was 0–28 °C and optimum growth occurred at 24 °C. The novel isolate does not require NaCl; growth was observed between 0 and 5 % NaCl with optimum growth at 0·5 % (w/v). The novel isolate was a catalase-negative chemoorganoheterotroph that used as substrates sugars and some products of proteolysis. The metabolic end products were acetate, ethanol and CO2. Strain FTR1T was sensitive to ampicillin, tetracycline, chloramphenicol, rifampicin, kanamycin and gentamicin. 16S rRNA gene sequence analysis showed 99·8 % similarity between strain FTR1T and Carnobacterium alterfunditum, but DNA–DNA hybridization between them demonstrated 39±1·5 % relatedness. On the basis of genotypic and phenotypic characteristics, it is proposed that strain FTR1T (=ATCC BAA-754T=JCM 12174T=CIP 108033T) be assigned to the novel species Carnobacterium pleistocenium sp. nov.

Four strains of an asexual arthroconidial yeast species were isolated from Drosophila flies in two Atlantic rain forest sites in Brazil and two strains from oak tasar silkworm larvae (Antheraea proylei) in India. Analysis of the sequences of the D1/D2 large subunit rRNA gene showed that this yeast represented a novel species of the genus Geotrichum, described as Geotrichum silvicola sp. nov. The novel species was related to the ascogenous genus Galactomyces. The closest relatives of Geotrichum silvicola were Galactomyces sp. strain NRRL Y-6418 and Galactomyces geotrichum. The type culture of Geotrichum silvicola is UFMG-354-2T (=CBS 9194T=NRRL Y-27641T).

During a survey of carotenogenic yeasts carried out in north-western Patagonia (Argentina), several ballistoconidia-producing strains belonging to the order Sporidiobolales were isolated from aquatic environments. Five strains were found to represent two novel species, for which the names Sporidiobolus longiusculus and Sporobolomyces patagonicus are proposed, with CBS 9654T (=PYCC 5818T=CRUB 1044T) and CBS 9657T (=PYCC 5817T=CRUB 1038T) as the type strains, respectively. The elongated basidia, which are five to six times longer that those of the remaining species of the genus Sporidiobolus, are a particular micromorphological feature of Sporidiobolus longiusculus. On the basis of the sequences of the D1/D2 domains of the 26S rRNA gene, the species most closely related to Sporidiobolus longiusculus is Sporobolomyces bannaensis, whereas Sporobolomyces marcillae is the closest relative of Sporobolomyces patagonicus. Complete internal transcribed spacer sequence analysis confirmed the separate position of Sporidiobolus longiusculus, whereas for Sporobolomyces patagonicus no nucleotide differences were found with respect to Sporidiobolus pararoseus CBS 491T. Negative mating experiments between strains of Sporobolomyces patagonicus and strains of Sporidiobolus pararoseus together with the low DNA–DNA reassociation values for the type strains of the two species validated the proposal of Sporobolomyces patagonicus as a distinct species. Information on additional Patagonian Sporobolomyces isolates is also included in this report.

One hundred and seventy-eight lactobacilli isolated from wine were characterized by a polyphasic approach. Strains were phenotypically identified at genus and species level by classical tests including the analysis of cell morphology, homo/heterofermentative character, sugar fermentation patterns, growth at different temperatures and the optical nature of the isomer of lactic acid produced from glucose. Molecular techniques such as random amplification of polymorphic DNA (RAPD), amplified 16S rDNA restriction analysis (16S-ARDRA), PFGE-RFLP and ribotyping were used to characterize strains, and their potential for identification and/or typing was evaluated. The information obtained with these techniques was processed with the BioNumerics software in order to analyse relationships existing between isolated strains and various reference species of the genus. Then, taxonomic dendrograms were obtained, and this information allowed the proposal of molecular procedures suitable for the identification and typing of these wine micro-organisms. The techniques useful for both identification and typing were RAPD and ribotyping, while 16S-ARDRA was only useful for identification and PFGE-RFLP only for typing purposes. The wine strains were identified as Lactobacillus brevis (19 strains), Lactobacillus collinoides (2 strains), Lactobacillus hilgardii (71 strains), Lactobacillus paracasei (13 strains), Lactobacillus pentosus (2 strains), Lactobacillus plantarum (34 strains) and Lactobacillus mali (10 strains).

A total of 128 strains was isolated from more than 23 legume hosts in Korea. Phylogenetic relationships between these Korean isolates and reference strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were analysed using their 16S rRNA gene and internally transcribed spacer (ITS) region sequences. Among the Bradyrhizobium strains, dendrograms based on both the 16S rRNA gene and ITS region sequences produced two main groups. The ITS tree yielded at least two new clusters that were discernable from the seven previously delineated genospecies. Large discrepancies were revealed between phylogenetic dendrograms based on 16S rRNA gene and ITS region sequences for members of the genus Rhizobium, reflecting their taxonomic heterogeneity. The amalgamation of Rhizobium and former members of Agrobacterium was confirmed using the 16S rRNA tree. Phylogenetic analysis of ITS region sequences showed that the Rhizobium giardinii clade (group II) and the Rhizobium radiobacter/Rhizobium rubi clade (group III) could be tentatively recognized as groups that are separable from the core group (group I), which includes Rhizobium leguminosarum. Dendrograms based on the 16S rRNA gene and ITS region sequences of Mesorhizobium strains were highly conflicting due to the poor taxonomic resolution of the 16S rRNA gene sequences and the low confidence in the ITS dendrogram. Several Korean isolates within the genus Mesorhizobium are thought to represent novel taxa when considering their relatively low ITS region sequence similarities (<80 %) to the reference strains.