- Volume 53, Issue 6, 2003

Two thermophilic, Gram-negative, non-spore-forming, multicellular filamentous micro-organisms were isolated from thermophilic granular sludge in an upflow anaerobic sludge blanket reactor treating fried soybean-curd manufacturing waste water (strain UNI-1T) and from a hot spring sulfur-turf in Japan (strain STL-6-O1T). The filaments were longer than 100 μm and of 0·2–0·3 μm (strain UNI-1T) or 0·7–0·8 μm (strain STL-6-O1T) in width. Strain UNI-1T was a strictly anaerobic organism. The optimum temperature for growth was around 55 °C; growth occurred in the range 50–60 °C. The optimum pH for growth was around 7·0; growth occurred in the range pH 6·0–8·0. Strain STL-6-O1T was a facultatively aerobic bacterium. The optimum temperature for growth was around 55 °C; growth occurred in the range 37–65 °C. The optimum pH for growth was around 7·5–8·0; growth occurred in the range pH 7·0–9·0. The two organisms grew chemo-organotrophically on a number of carbohydrates and amino acids in the presence of yeast extract. The G+C content of the DNA of strains UNI-1T and STL-6-O1T was 54·5 and 59·0 mol%, respectively. Major cellular fatty acids for strain UNI-1T were C16 : 0, C15 : 0, C14 : 0 and C18 : 0, whereas those for strain STL-6-O1T were C18 : 0, C16 : 0, C17 : 0 and iso-C17 : 0. MK-10 was the major quinone from aerobically grown STL-6-O1T cells. Phylogenetic analyses based on 16S rDNA sequences revealed that both strains belong to an uncultured, previously recognized clone lineage of the phylum Chloroflexi (formerly known as green non-sulfur bacteria). These phenotypic and genetic properties suggested that each strain should be classified into a new independent genus; hence, the names Anaerolinea thermophila and Caldilinea aerophila are proposed for strains UNI-1T (=JCM 11387T=DSM 14523T) and STL-6-O1T(=JCM 11388T=DSM 14525T), respectively. These strains represent the type and sole species of the genera Anaerolinea and Caldilinea, respectively.

In addition to rice yellow dwarf (RYD) phytoplasma, several phytoplasmas infect gramineous plants, including rice orange leaf, bermuda grass white leaf, brachiaria grass white leaf and sugarcane white leaf phytoplasmas. To investigate whether the RYD phytoplasma is a discrete, species-level taxon, several isolates of the aforementioned phytoplasmas were analysed using PCR-amplified 16S rDNA sequences. Two RYD isolates, RYD-JT and RYD-Th, were almost identical (99·2 %), but were distinct (similarities of 96·3–97·9 %) from other phytoplasma isolates of the RYD 16S-group. The notion that the RYD phytoplasma constitutes a unique taxon is also supported by its unique insect vector (Nephotettix sp.), its unique host plant in nature (rice) and its limited geographical distribution (Asia). In Southern blot analysis, chromosomal and extrachromosomal DNA probes of the RYD phytoplasma reportedly did not hybridize with those of closely related phytoplasmas. These properties of the RYD phytoplasma clearly indicate that it represents a novel taxon, ‘Candidatus Phytoplasma oryzae’.

Six strains of anaerobic, Gram-negative coccobacilli isolated from the root canals of patients with endodontic infections (five strains) and from a deep periodontal pocket (one strain) were subjected to a comprehensive range of phenotypic and genetic tests and were found to comprise a homogeneous group. Following 16S rRNA gene sequence analysis, they were found to be most closely related to Dialister pneumosintes, with 93 % sequence similarity between the two taxa. A novel species, Dialister invisus sp. nov., is proposed. Biochemically, the species is largely unreactive and is asaccharolytic, with only traces of acetate and propionate detected as metabolic end-products. The G+C content of the DNA of D. invisus strains is 45–46 mol%. The type strain is E7.25T (=CCUG 47026T=DSM 15470T).

A novel, extremely thermophilic bacterium, designated strain 17ST, was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan. The cells were rods with no apparent motility, most of which were narrow in the middle in the exponential-growth phase and had several polar flagella at both ends. Growth was observed between 45 and 80 °C (optimum temperature, 70–75 °C; doubling time, 80 min) and between pH 5·0 and 7·0 (optimum pH, 5·4). The isolate was a strictly anaerobic chemolithoautotroph that was capable of using molecular hydrogen as its sole energy source and carbon dioxide as its sole carbon source. Elemental sulfur (S0) was required for growth as an electron acceptor. The G+C content of the genomic DNA was 34·6 mol%. Phylogenetic analysis based on 16S rDNA sequences indicated that the isolate was related to Thermovibrio ruber ED11/3LLKT and Desulfurobacterium thermolithotrophum BSAT, whilst it appeared to be a novel lineage prior to the divergence of these genera. This isolate could also be differentiated from both T. ruber ED11/3LLKT and D. thermolithotrophum BSAT on the basis of physiological properties. The name Balnearium lithotrophicum gen. nov., sp. nov. is proposed for this isolate (type strain, 17ST=JCM 11970T=ATCC BAA-736T).

The taxonomic position of four heterotrophic, aerobic, Gram-negative, non-motile and moderately halophilic marine bacteria, isolated from the green alga Acrosiphonia sonderi (Kütz) Kornm, was established. 16S rDNA sequence analysis indicated that the strains studied are members of the family Flavobacteriaceae, in which they form a distinct lineage. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, the novel bacteria were classified as Mesonia algae gen. nov., sp. nov. The type strain is KMM 3909T (=KCTC 12089T=CCUG 47092T).

Strain ALEN 2T was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic. It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite. It was moderately halophilic and obligately alkaliphilic. On the basis of genetic analysis and its unique phenotype, strain ALEN 2T (=DSM 14787T=UNIQEM 213T) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.

A novel mesophilic, sulfur- and thiosulfate-oxidizing bacterium, strain OK10T, was isolated from deep-sea sediments at the Hatoma Knoll in the Mid-Okinawa Trough hydrothermal field. Cells of strain OK10T were short rods, each being motile by means of a single polar flagellum. The isolate grew at 10–40 °C (optimum 25 °C) and pH 4·5–9·0 (optimum pH 6·5). It grew chemolithoautotrophically with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Molecular hydrogen did not support growth. The G+C content of the genomic DNA of strain OK10T was 35·2 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolate belonged to the ε-Proteobacteria. On the basis of its physiological and molecular characteristics, strain OK10T (=ATCC BAA-671T=JCM 11897T) represents the sole species of a new genus, Sulfurimonas, for which the name Sulfurimonas autotrophica is proposed.

A marine bacterium producing protease inhibitors was isolated from neritic sea water and was studied phenotypically, genotypically and phylogenetically. This bacterium (strain B-10-31T) produced three types of protease inhibitor, namely, marinostatin, monastatin and leupeptin, which were considerably different in terms of their chemical structure and properties. Strain B-10-31T was a rod-shaped, non-spore-forming, Gram-negative, strictly aerobic bacterium that was motile by means of one polar flagellum. The strain required Na+ for growth and exhibited optimal growth at 27 °C, pH 8·0 and 2 % (w/v) NaCl. It utilized various substrates, such as d-glucose, maltose, maltotriose, N-acetylglucosamine, l-threonine, l-serine, l-arginine, l-proline, l-α-alanine and l-glutamate, as the sole energy source. Ubiquinone-8 was the major respiratory quinone. The major fatty acids were C16 : 0, C16 : 1 ω7c, C16 : 1 ω9c and C18 : 1 ω7c. The G+C content of the DNA of strain B-10-31T was 42·0 mol%. Phylogenetic analysis, based on 16S rDNA sequences, showed that the strain clustered in the γ-Proteobacteria. The aerobic marine bacterium Pseudoalteromonas bacteriolytica was the species most closely related to the new isolate (90·4 % 16S rDNA sequence similarity); other described species in the γ-Proteobacteria cluster showed low levels of sequence similarity with strain B-10-31T (<90 %). Based on the above results, it is proposed that the novel marine bacterium should be classified as a new species, for which the name Pseudoalteromonas sagamiensis (type strain B-10-31T=JCM 11461T=DSM 14643T) is proposed.

Five alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the gut of abalones Haliotis laevigata and Haliotis rubra. Phylogenetic analyses based on 16S rDNA data indicated that these strains are related closely to Vibrio halioticoli (98 % 16S rDNA sequence similarity). DNA–DNA hybridization and fluorescent amplified fragment length polymorphism fingerprinting demonstrated that the five strains constituted a single species that was different from all currently known vibrios. The name Vibrio superstes sp. nov. (type strain, LMG 21323T=IAM 15009T=G3-29T; DNA G+C content, 48·0–48·9 mol%) is proposed to encompass this novel taxon. Several phenotypic features were disclosed that discriminate V. superstes from other Vibrio species: V. superstes sp. nov. and V. halioticoli can be differentiated on the basis of 17 traits (indole production, β-galactosidase test and assimilation of 15 carbon compounds).

A polyphasic taxonomic study (employing whole-cell protein and fatty acid analyses, 16S rDNA sequencing, DNA–DNA hybridization, determination of DNA G+C content, antibiotic susceptibility testing and extensive phenotypic characterization) was performed on 10 isolates that appeared to be related to Alcaligenes faecalis. The isolates were recovered from diverse environments that included human clinical samples. 16S rDNA sequence analysis indicated that these isolates belonged to the genus Achromobacter. Whole-cell protein analysis distinguished two groups, which were confirmed by DNA–DNA hybridization. Based on the results of this study, the organisms were classified as two novel Achromobacter species, Achromobacter insolitus sp. nov. (type strain, LMG 6003T) and Achromobacter spanius sp. nov. (type strain, LMG 5911T). Achromobacter insolitus can be distinguished from Achromobacter spanius by its ability to grow on acetamide and to assimilate mesaconate and aconitate, and by its inability to assimilate diaminobutane. Various tests allow the differentiation of both novel species from other Achromobacter species, including growth on acetamide, denitrification and assimilation of d-glucose, d-xylose, mesaconate, aconitate and diaminobutane.

A polyphasic taxonomic study was performed on nine isolates recovered from various human clinical samples. Phenotypically, these isolates resembled Alcaligenes faecalis. Whole-cell protein analysis distinguished two different species, and this was confirmed by DNA–DNA hybridizations. Cellular fatty acid analysis and 16S rDNA sequence analysis indicated that these isolates were related to the genera Alcaligenes, Bordetella, Achromobacter and Pigmentiphaga and belonged to the family Alcaligenaceae. On the basis of the results of this study, the organisms were classified in a novel genus, Kerstersia gen. nov. This genus comprises one species, Kerstersia gyiorum sp. nov. (type strain LMG 5906T=API 184-2-84T=CCUG 47000T), and several unnamed isolates. The DNA G+C content of members of the genus Kerstersia is between 61·5 and 62·9 mol%. On the basis of previously published DNA–DNA hybridization results and data from chemotaxonomic studies, it is proposed that Alcaligenes denitrificans Rüger and Tan 1983 be reclassified as Achromobacter denitrificans comb. nov.

Three brownish-yellow bacterial strains were isolated from the western Sargasso Sea by high-throughput culturing methods and characterized by polyphasic approaches. All isolates were Gram-negative, strictly aerobic, chemoheterotrophic, non-motile short rods that contained carotenoid pigments. Phylogenetic analyses based on 16S rRNA gene sequences, DNA–DNA hybridization and DNA G+C content, along with phenotypic characteristics, revealed that they belonged to the same species. The strains utilized a wide range of substrates, including pentoses, hexoses, oligosaccharides, sugar alcohols, organic acids and amino acids, as sole carbon sources. The DNA G+C content of the isolates ranged from 57·6 to 59·9 mol%. The predominant cellular fatty acid constituent was C18 : 1 ω7c, whilst C16 : 0, C18 : 0 and C19 : 0 ω8c cyclo were also abundant. The organism related most closely to these strains, as determined by 16S rDNA sequence comparison, was the recently described species Aurantimonas coralicida (93·3–93·8 % similarity). Phylogenetic analyses indicated that the strains formed a distinct and deep evolutionary lineage of descent, together with A. coralicida, within the order ‘Rhizobiales’ of the α-Proteobacteria. This lineage could not be associated with any of the ten known families in the order ‘Rhizobiales’. From polyphasic evidence, it is proposed that the strains be placed into a novel genus and species, Fulvimarina pelagi gen. nov., sp. nov. (type strain, HTCC2506T=ATCC BAA-666T=KCTC 12091T=DSM 15513T).

During an investigation of bacterial diversity at hydrothermal vents on the Lō‘ihi Seamount, Hawai‘i, a novel bacterium (designated L2-TRT) was cultivated, which shares 99·9 % 16S rRNA gene sequence similarity over 1415 nt with an uncultured eubacterium from sediment at a depth of 11 000 m in the Mariana Trench. The nearest cultivated neighbour of L2-TRT, however, is Idiomarina abyssalis KMM 227T, with which it shares 98·9 % 16S rRNA sequence similarity. L2-TRT differed from I. abyssalis KMM 227T in several phenotypic respects, including growth at 46 °C and in medium that contained 20 % (w/v) NaCl. DNA–DNA hybridization data showed that L2-TRT did not belong to the species I. abyssalis (43·4 % DNA–DNA reassociation). Cells of L2-TRT were Gram-negative rods, 0·35 μm wide and 0·7–1·0 μm long, which were occasionally up to 1·8 μm in length. Cells were motile by a single polar or subpolar flagellum. The major fatty acid in L2-TRT was iso-C15 : 0 (32·6 %). The DNA G+C content was 47·4 mol%. Phenotypic and genotypic analyses indicated that L2-TRT could be assigned to the genus Idiomarina but, based on significant phenotypic and genotypic differences, constituted a novel species within this genus, Idiomarina loihiensis sp. nov., of which L2-TRT (=ATCC BAA-735T=DSM 15497T) is the type strain.

A polyphasic taxonomic study was performed to characterize a new bacterial isolate, designated KMM 3654T, from a marine bottom sand sample. The strain was Gram-negative, encapsulated, aerobic, moderately halophilic and grew between 0·5 and 10 % NaCl and at 4–42 °C. Its DNA G+C content was 56·4 mol%. Isolate KMM 3654T was phylogenetically closely related to members of the genus Oceanimonas, showing 96·7 and 95·6 % 16S rRNA gene sequence similarity to Oceanimonas doudoroffii DSM 7028T and Oceanimonas baumannii ATCC 700832T, respectively. Strain KMM 3654T shared some physiological and chemotaxonomic properties with these two Oceanimonas species, but differed from them in morphology, growth at 4 °C, urease activity, weak phenol degradation and utilization of phenylacetate. On the basis of phenotypic and phylogenetic evidence, Oceanisphaera litoralis gen. nov., sp. nov. is proposed, with the type strain KMM 3654T (=DSM 15406T).

Five bacterial strains were isolated from a non-toxigenic strain of the marine dinoflagellate Alexandrium tamarense (Lebour) Balech CCMP 116 (NEPCC C116), during a survey of the diversity of bacteria associated with paralytic shellfish toxin-producing cultures of Alexandrium spp. (Dinophyta). All strains were strictly aerobic, Gram-negative, straight or curved rods. Cells were dimorphic, with stalks (or prosthecae) and non-motile or non-stalked and motile, by means of a single polar flagellum. The bacteria grew best at salt concentrations ranging from 2 to 10 % and growth occurred at 10 °C, but not at 50 °C. The G+C content of the chromosomal DNA of the strains was determined to be 61–62 mol%. Major cellular fatty acids of the bacteria presented a unique profile. 16S rRNA gene sequence analysis showed the five strains to be related to genera of budding bacteria of marine origin in the ‘Alphaproteobacteria’, namely, Hirschia, Maricaulis and Hyphomonas, although they exhibited substantial differences in morphology, substrate utilization and fatty acid profile to members of these genera. The five strains are proposed to comprise a new species of a new genus, Oceanicaulis alexandrii gen. nov., sp. nov., the type strain of which is C116-18T (=DSM 11625T=NCIMB 13905T).

Previously, 22 aerobic Gram-negative bacteria were isolated from biofilms growing on granules of the synthetic polyester poly(ε-caprolactone); the granules were used as a fixed bed in a denitrification reactor. All the strains showed similar fatty acid profiles. The 16S rRNA gene sequences of five strains were phylogenetically related to Thermomonas spp. Repetitive extragenic palindromic DNA-PCR (REP-PCR) fingerprinting revealed four groups, and DNA hybridizations between representative strains showed that the strains belonged to two new species within the genus Thermomonas, for which the names Thermomonas fusca (type strain LMG 21737T=DSM 15424T) and Thermomonas brevis (type strain LMG 21746T=DSM 15422T) are proposed. Both species are able to grow at low temperatures, but not at 50 °C, and are non-haemolytic. Both species can be differentiated by several other phenotypic features from earlier described species of the genus Thermomonas. Cell extracts contain mainly branched fatty acids, with C15 : 0 iso, C17 : 1 iso ω9c, C11 : 0 iso 3OH and C11 : 0 iso as main constituents. The G+C content of the DNA of the novel species is between 67·6 and 68·7 mol%.

An aerobic, Gram-negative, non-fermentative, rod-shaped, motile, non-pigmented bacterium, KMM 1406T, was isolated from a sample of Pacific deep sea water and investigated for phenotypic characteristics, chemotaxonomic features and phylogenetic relationships. The deep-sea isolate exhibited growth in 0–8 % (w/v) NaCl and at 4–37 °C, hydrolytic activity on gelatin, Tween 80 and starch and lack of d-glucose utilization. The major fatty acids were C16 : 0, C16 : 1 ω9c, C17 : 1 ω8c and C18 : 1 ω7c. The DNA G+C content was 49·6 mol%. 16S rRNA gene sequence analysis revealed that strain KMM 1406T was related closely to Rheinheimera baltica DSM 14885T within the γ-Proteobacteria, with 96·8 % sequence similarity. On the basis of phenotypic and molecular data, a novel species, Rheinheimera pacifica sp. nov., is proposed. The type strain is KMM 1406T (=IAM 15043T=JCM 12090T=NRIC 0539T=CCUG 46544T).