- Volume 58, Issue 11, 2008

Five Gram-negative, rod-shaped, moderately halophilic and denitrifying strains, designated XK1T, XK2, XK3, XK4 and XK5, were isolated from a saline and alkaline soil in Korla, north-western China. These isolates could grow anaerobically using either nitrate or nitrite as terminal electron acceptors and produced gas from nitrate vigorously. A comparison and phylogenetic analysis of 16S rRNA gene sequences placed these isolates in the genus Halomonas within the family Halomonadaceae. The isolates shared the highest 16S rRNA gene sequence similarities with Halomonas ventosae Al12T (95.6 %), Halomonas alimentaria YKJ-16T (95.5 %) and Halomonas shengliensis SL014B-85T (95.2 %) (values determined by mega 3.1; direct comparison results with GenBank were even lower, not ≥94 %). Sequence similarities with other recognized species were below 95.0 %, far below the 97.0 % threshold generally accepted for the delineation of separate species. BOX-PCR fingerprinting and DNA–DNA hybridization showed high similarities among the five strains which indicated they were members of the same species. The major fatty acids were C18 : 1 ω8t, C16 : 0 and C18 : 1 ω7t. The DNA G+C content was 65.3 mol%. All the results of the phenotypic and genetic analyses supported the hypothesis that the five new strains represent a novel species within the genus Halomonas, for which the name Halomonas korlensis sp. nov. is proposed. The type strain is XK1T (=CGMCC 1.6981T=DSM 19633T).

Thirty-five isolates obtained from a marine electroactive biofilm grown on a stainless steel cathode (Genoa, Italy) were investigated by a polyphasic taxonomic approach. Whole-cell fatty acid methyl ester analysis indicated that the isolates belonged to the class Gammaproteobacteria and were closely related or belonging to the genus Alteromonas. Genomic fingerprinting using the BOX-PCR primer delineated five clusters of isolates with similar BOX-PCR fingerprints. This study demonstrated that isolates from four of the BOX-PCR clusters belonged to Alteromonas macleodii and that the 14 isolates representing BOX-PCR cluster 1 constituted a novel species, which shared 98.4 % 16S rRNA gene sequence similarity with its nearest phylogenetic neighbour, Alteromonas hispanica. Both phenotypic and genotypic analyses enabled this novel species, for which the name Alteromonas genovensis sp. nov. is proposed, to be differentiated from established species of the genus Alteromonas. The DNA G+C content of Alteromonas genovensis sp. nov. is 44.5 mol% and the type strain is LMG 24078T (=CCUG 55340T).

The taxonomic relatedness between the species Myxococcus flavescens and Myxococcus virescens was investigated. Literature data had already indicated the synonymy between the two species but this observation had not been formalized. Additional evidence that the two taxa represent a single species was provided by comparison of metabolic properties, cellular fatty acid profiles and from a DNA–DNA reassociation value of >80 %. Data from this study led to the proposal that M. flavescens should be reclassified as a later synonym of M. virescens.

Twenty-four Mesorhizobium strains were isolated from desert soils in the Xinjiang region of China and were characterized by a polyphasic approach. These strains grouped into three clusters in IGS-RFLP, SDS-PAGE analysis of whole-cell proteins and BOX-PCR analysis, corresponding to genomic species V, VI and VII as found in a previous study. The results were supported by sequencing analyses of rrs, IGS, atpD and recA genes. Genospecies VII was most related to Mesorhizobium septentrionale, while genospecies V and VI were both most closely related to Mesorhizobium tianshanense, but were distinct from each other and from M. tianshanense. The DNA–DNA hybridization value between the representative strain CCBAU 83284 (genospecies VII) and the type strain of M. septentrionale was 90.1 %. Genospecies VII was thus defined as M. septentrionale. The DNA–DNA relatedness value for representative strains of genospecies V or VI with the related reference strains of recognized species were always lower than 60 %. Low values of DNA–DNA hybridization (32.79 %) between representative strains of genospecies V (CCBAU 83330T) and of VI (CCBAU 83306T) were also observed. Based upon these results, two novel species are proposed: Mesorhizobium gobiense sp. nov. represented by genospecies V (type strain, CCBAU 83330T=LMG 23949T=HAMBI 2974T) and Mesorhizobium tarimense sp. nov. represented by genospecies VI (type strain, CCBAU 83306T=LMG 24338T=HAMBI 2973T). Strain CCBAU 83278 grouped as the most peripheral member with genospecies VI in SDS-PAGE of whole-cell proteins and BOX-PCR analysis and in the phylogenetic tree of 16S-23S rRNA intergenic spacer (IGS) sequences. The results of analyses of rrs, atpD and recA gene sequences, as well as those of DNA–DNA hybridization studies, strongly supported the suggestion that this strain belonged to a species quite different from genospecies V and VI and from any other recognized species of the genus Mesorhizobium. As only one strain has been isolated to date, strain CCBAU 83278 was not proposed as a novel species in this study. Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov. could be differentiated from each other as well as from recognized species of the genus Mesorhizobium on the basis of phenotypic characteristics. The symbiotic loci (nodC and nifH) of the two novel species formed two phylogenetic branches related to Mesorhizobium loti and M. tianshanense. The type strains of the two novel species were able to nodulate Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra and Robinia pseudoacacia but not Astragalus membranaceus, Leucaena leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.

Five rhizobial strains representing the largest group in the genus Mesorhizobium associated with Caragana spp. in China were characterized taxonomically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these microsymbionts belonged to the genus Mesorhizobium, with Mesorhizobium tianshanense USDA 3592T, Mesorhizobium temperatum SDW018T and Mesorhizobium mediterraneum UPM-Ca36T as the closest neighbours (≥99.5 % 16S rRNA gene sequence similarity). Genotypic fingerprinting by whole-cell protein electrophoresis, DNA–DNA hybridization, comparative housekeeping sequence analysis of the atpD, glnII and recA genes, fatty acid profiles and a series of phenotypic and physiological tests allowed the novel group to be differentiated from all previously recognized species of the genus Mesorhizobium. This group therefore represents a novel species, for which the name Mesorhizobium caraganae sp. nov. is proposed with the type strain CCBAU 11299T (=LMG 24397T=HAMBI 2990T). Cross-inoculation tests showed that strain CCBAU 11299T could form effective nodules on Caragana microphylla, Caragana intermedia, Glycyrrhiza uralensis, Astragalus adsurgens and Phaseolus vulgaris.

A coccoid, phototrophic purple sulfur bacterium was isolated in pure culture from a mud sample collected from brackish water in the Bhitarkanika mangrove forest of Orissa, India, in a medium containing 2 % NaCl (w/v). This bacterium, strain JA304T, was Gram-negative and had a requirement for NaCl. Intracellular photosynthetic membranes were of the vesicular type. The colour of the phototrophically grown culture was saddle-brown. Bacteriochlorophyll a and the carotenoid lycopene were present as photosynthetic pigments. Strain JA304T was able to grow photolithoautotrophically and could photoassimilate a number of organic substrates. Yeast extract was required for growth of strain JA304T. The DNA G+C content was 68.1–68.9 mol%. 16S rRNA gene sequence comparisons indicate that the isolate represents a member of the Chromatiaceae within the class Gammaproteobacteria. According to sequence comparison data, strain JA304T is positioned distinctly outside the group formed by the four genera Thiocystis, Chromatium, Allochromatium and Thermochromatium, with only 86.7–91.0 % sequence similarity. Distinct morphological, physiological and genotypic differences from these previously described taxa support the classification of this isolate as a representative of a novel species in a new genus, for which the name Thiophaeococcus mangrovi gen. nov., sp. nov. is proposed. The type strain of Thiophaeococcus mangrovi is JA304T (=JCM 14889T =DSM 19863T).

Ascogenous yeasts were isolated from a decaying mangosteen fruit (Garcinia mangostana L., Clusiaceae). Based on colony morphology and RAPD analysis, the strains were grouped into four groups (G-I, G-II, G-III and G-IV). Phenotypic properties and analysis of the D1/D2 domain sequence of the 26S rRNA gene identified representative members of groups G-II, G-III and G-IV as Pichia manshurica (=Pichia galeiformis), Pichia fermentans and Pichia anomala, respectively. Representatives of group G-I, YS110T and YS111, showed similar phenotypic traits, 99.9 % similarity in the sequence of the D1/D2 domain of the 26S rRNA gene and ITS1–5.8S rRNA gene–ITS2 sequence and 92 % DNA–DNA relatedness. Hence, YS110T and YS111 are regarded as members of the same species. Based on D1/D2 domain and ITS region sequences, the nearest phylogenetic neighbours of YS110T and YS111 were identified as Pichia membranifaciens and P. manshurica. However, YS110T differs from these two nearest phylogenetic neighbours by >3 % in the D1/D2 domain sequence and by >18 % with respect to the ITS region sequence. In addition, YS110T and YS111 differ from P. membranifaciens NRRL Y-2026T and P. manshurica NRRL Y-27978T with respect to a number of phenotypic traits. The strains show 52–57 % DNA–DNA relatedness with P. membranifaciens NRRL Y-2026T and P. manshurica NRRL Y-27978T. Strains YS110T and YS111 are proposed as two strains of a novel species, for which the name Pichia garciniae sp. nov. is proposed. The type strain is YS110T (=NRRL Y-48422T =CBS 10758T).

A novel strictly anaerobic, moderately thermophilic, sulfate-reducing bacterium, designated strain Lam5T, was isolated from a hot spring in north-east Tunisia and was characterized phenotypically and phylogenetically. The isolate stained Gram-negative but had a Gram-positive-type cell wall. The strain comprised endospore-forming, slightly curved rod-shaped cells with peritrichous flagella. It did not possess desulfoviridin. Strain Lam5T grew anaerobically at 40–60 °C (optimally at 55 °C) and at pH 5.8–8.2 (optimally at pH 7.1); it did not require NaCl but tolerated concentrations up to 1.5 % (w/v). It utilized lactate, pyruvate, formate, ethanol, butanol, glycerol, propanol and H2 (plus acetate) as electron donors. Lactate was oxidized and pyruvate was fermented to acetate. Sulfate, sulfite, thiosulfate, As(V) and Fe(III) (but not elemental sulfur, fumarate, nitrate or nitrite) were used as electron acceptors. The G+C content of the genomic DNA was 46.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain Lam5T was a member of the genus Desulfotomaculum, with Desulfotomaculum putei as its closest relative (96 % similarity to the type strain). On the basis of genotypic, phenotypic and phylogenetic data, strain Lam5T represents a novel species of the genus Desulfotomaculum, for which the name Desulfotomaculum hydrothermale sp. nov. is proposed. The type strain is Lam5T (=DSM 18033T =JCM 13992T).

Three Gram-positive, catalase-negative, motile, rod-shaped bacteria were isolated from fermented stinky tofu brine. These strains, designated YIT 11306T, YIT 11317 and YIT 11318, were discriminated from five isolates on the basis of randomly amplified polymorphic DNA profiles. They produced l-lactic acid as the main end product from glucose without gas formation, synthesized dextran from sucrose and hydrolysed aesculin. Ammonia was not produced from arginine. Comparative 16S rRNA gene sequence analysis demonstrated that the novel isolates were members of the genus Lactobacillus. Based on levels of 16S rRNA gene sequence similarity, the three novel strains were related most closely to the type strains of Lactobacillus mali (97.2 %) and Lactobacillus satsumensis (96.8 %). However, levels of DNA–DNA relatedness between the novel isolates and the type strains of L. mali and L. satsumensis were less than 10 %. The phenotypic and genotypic data demonstrate that the three strains represent a single novel species of the genus Lactobacillus, for which the name Lactobacillus capillatus sp. nov. is proposed. The type strain is YIT 11306T (=JCM 15044T=BCRC 17811T=DSM 19910T).

A bacterial strain, designated Ch-32T, was isolated from the phyllosphere of Castanea sativa in Spain. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus within the same subgroup as Paenibacillus xinjiangensis and Paenibacillus glycanilyticus, with similarities of 96.3 and 96.8 %, respectively. DNA–DNA hybridization values for strain Ch-32T with these two species were lower than 20 %. The novel isolate was a Gram-variable, motile, sporulating rod. It produced catalase and oxidase and hydrolysed cellulose, gelatin and aesculin. Acetoin and urease production, nitrate reduction and starch hydrolysis were negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 were the major fatty acids. The DNA G+C content was 46 mol%. Phylogenetic, DNA relatedness and phenotypic analyses showed that strain Ch-32T should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus castaneae sp. nov. is proposed; the type strain is Ch-32T (=CECT 7279T=DSM 19417T).

Several strains of heterotrophic, anaerobic thermophilic bacteria were isolated from hot springs of the Uzon Caldera, Kamchatka, Far East Russia. Strain JW/IW010T was isolated from a hot spring within the West sector of the Eastern Thermal field, near Pulsating Spring in the Winding Creek area. Cells of strain JW/IW010T were straight to slightly curved rods, 0.5 μm in width and variable in length from 2 to 5 μm and occasionally up to 15 μm, and formed oval subterminal spores. Cells stained Gram-negative, but were Gram-type positive. Growth was observed between 32.5 and 69 °C with an optimum around 61 °C (no growth occurred at or below 30 °C, or at or above 72 °C). The pH60 °C range for growth was 4.2–8.9 with an optimum at 7.1 (no growth occurred at or below pH60 °C 3.9, or at 9.2 or above). The shortest observed doubling-time at pH60 °C 6.9 and 61 °C was 30 min. Strain JW/IW010T was chemo-organotrophic; yeast extract, peptone, Casamino acids and tryptone supported growth. Yeast extract was necessary for the utilization of non-proteinaceous substrates, and growth was observed with inulin, cellobiose, maltose, sucrose, glucose, fructose, galactose, mannose, xylose, trehalose, mannitol, pyruvate and crotonate. The G+C content of the genomic DNA of strain JW/IW010T was 33.6 mol% (HPLC method). The major phospholipid fatty acids were iso-15 : 0 (53.5 %), 15 : 0 (11.8 %), 16 : 0 (7.3 %), 10-methyl 16 : 0 (7.3 %) and anteiso-15 : 0 (5.3 %). 16S rRNA gene sequence analysis placed strain JW/IW010T in the genus Thermoanaerobacter of the family ‘Thermoanaerobacteriaceae’ (Firmicutes), with Thermoanaerobacter sulfurigignens JW/SL-NZ826T (97 % 16S rRNA gene sequence similarity) and Thermoanaerobacter kivui DSM 2030T (94.5 %) as the closest phylogenetic relatives with validly published names. The level of DNA–DNA relatedness between strain JW/IW010T and Thermoanaerobacter sulfurigignens JW/SL-NZ826T was 64 %. Based on the physiological, phylogenetic and genotypic data, strain JW/IW010T represents a novel taxon, for which the name Thermoanaerobacter uzonensis sp. nov. is proposed. The type strain is JW/IW010T (=ATCC BAA-1464T=DSM 18761T). The effectively published strain, 1501/60, of ‘Clostridium uzonii’ [ Krivenko, V. V., Vadachloriya, R. M., Chermykh, N. A., Mityushina, L. L. & Krasilnikova, E. N. (1990). Microbiology (English translation of Mikrobiologiia) 59, 741–748 ] had approximately 88.0 % DNA–DNA relatedness with strain JW/IW010T and was included in the novel taxon.

A Gram-positive, alkaliphilic bacterium, designated strain BA288T, was isolated from sandy soil. Cells were facultatively anaerobic, endospore-forming rods that were motile by means of peritrichous flagella. The strain grew at 15–40 °C and pH 7.0–11.0 (optimally at 30 °C and pH 9.0–9.5) and at salinities of 0–4 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain BA288T belonged to the genus Bacillus and that Bacillus herbersteinensis D-1,5aT, Bacillus humi LMG 22167T, Bacillus cohnii DSM 6307T and Bacillus litoralis SW-211T were the closest neighbours (96.2, 96.0, 96.0 and 95.9 % sequence similarity, respectively). The genomic DNA G+C content was 37.9 mol% and the predominant menaquinone was MK-7. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and iso-C14 : 0. The peptidoglycan type was A1γ (meso-diaminopimelic acid). Therefore, on the basis of phylogenetic, phenotypic and chemotaxonomic properties, strain BA288T represents a novel species of the genus Bacillus, for which the name Bacillus alkalitelluris sp. nov. is proposed. The type strain is BA288T (=KCTC 3947T =DSM 16976T).

Among a large collection of Taiwanese soil isolates, a novel Gram-variable, rod-shaped, motile, endospore-forming bacterial strain, strain V10537T, was subjected to a polyphasic study including 16S rRNA and gyrB gene sequence analysis, DNA–DNA hybridization experiments, cell wall peptidoglycan type, cellular fatty acid composition analysis and comparative phenotypic characterization. 16S rRNA gene sequence analysis indicated that the organism belonged to the genus Paenibacillus. Strain V10537T possessed meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. It contained menaquinone MK-7 as the predominant isoprenoid quinone and anteiso-C15 : 0 (53.6 %) and C16 : 0 (19.0 %) as the major fatty acids. Phylogenetically, the most closely related species to strain V10537T were Paenibacillus pabuli, Paenibacillus xylanilyticus, Paenibacillus amylolyticus, Paenibacillus barcinonensis and Paenibacillus illinoisensis, with 16S rRNA gene sequence similarities of 99.5, 98.8, 98.3, 98.2 and 98.1 % to the respective type strains. The gyrB gene sequence similarities between strain V10537T and these strains were 76.9–85.0 %. DNA–DNA hybridization experiments showed levels of relatedness of 8.5–45.6 % between strain V10537T and these strains. The DNA G+C content of strain V10537T was 46.7 mol%. Strain V10537T was clearly distinguishable from other Paenibacillus species and thus represents a novel species of the genus Paenibacillus, for which the name Paenibacillus taichungensis sp. nov. is proposed. The type strain is V10537T (=BCRC 17757T =DSM 19942T).