- Volume 56, Issue 3, 2006

A total of 52 strains of moderately halophilic bacteria isolated from hypersaline sediment of Lake Tebenquiche on the Atacama Saltern, Chile, were subjected to a taxonomic study. The morphological, physiological, biochemical and nutritional characteristics of the strains matched those described for the genus Chromohalobacter. Cells were Gram-negative, non-spore-forming, rod-shaped and motile. A black pigmentation was produced. One strain, designated LTS-4NT, grew optimally at 32 °C. Growth occurred in media containing 0·5–25 % (w/v) total salts; the optimum was 7·5 % (w/v) total salts. The pH range for growth was 5–10. The G+C content of the DNA of strain LTS-4NT was 59·8 mol%. Analysis of 16S rRNA gene sequence similarity revealed that strain LTS-4NT was closely related to Chromohalobacter species; however, DNA–DNA hybridization of representative strain LTS-4NT failed to associate the strain with any species of the genus Chromohalobacter with validly published names. Therefore, the name Chromohalobacter nigrandesensis sp. nov. is proposed. The type strain is LTS-4NT (=CECT 5315T=DSM 14323T).

Two moderately halotolerant Gram-negative bacteria were isolated from tidal flat sediment of the South Sea in Korea (the Korea Strait). The strains, designated M9T and M18T, were strictly aerobic, rod-shaped and non-spore-forming and motile with a flagellum and their major fatty acids were C16 : 0 and C19 : 0 cyclo ω8c. Strains M9T and M18T could grow in the presence of up to 13–15 % (w/v) NaCl, but their optimum salt concentrations were relatively low (0–3 %, w/v). The major predominant isoprenoid quinone was Q-8 and the G+C content of the genomic DNA was 57–58 mol%. Phylogenetic analyses and comparative 16S rRNA gene sequence studies revealed that strains M9T and M18T formed a phylogenetic lineage distinct from the genus Teredinibacter within the class Gammaproteobacteria and were most closely related to the genera Microbulbifer, Saccharophagus and Teredinibacter, with less than 92·5 % 16S rRNA gene sequence similarity. The level of 16S rRNA gene sequence similarity between the two strains was 96·7 %. On the basis of physiological and phylogenetic properties, strains M9T and M18T represent separate species within a novel genus of the class Gammaproteobacteria, for which the names Marinimicrobium koreense gen. nov., sp. nov. (type species) and Marinimicrobium agarilyticum sp. nov. are proposed. The type strains of Marinimicrobium koreense and Marinimicrobium agarilyticum are M9T (=KCTC 12356T=DSM 16974T) and M18T (=KCTC 12357T=DSM 16975T), respectively.

Three strains, 4M1T, 4M9 and 4M12, were isolated from cotton waste composts. These strains are Gram-negative, aerobic and non-spore-forming rods. 16S rRNA gene sequence comparisons demonstrated that these isolates were clustered phylogenetically within the genus Pseudoxanthomonas and 4M1T revealed sequence similarity levels of 96·9–99·0 % to six Pseudoxanthomonas species with validly published names. According to DNA–DNA hybridization, relatedness values between 4M1T and six known Pseudoxanthomonas species were in the range of 52–63 %. The DNA G+C content of the strains was 66·6–68·4 mol%. For a more detailed characterization of these strains, the physiological, chemotaxonomic and genotypic properties were evaluated. From the results of this study, the name Pseudoxanthomonas suwonensis sp. nov. is proposed, with the type strain 4M1T (=KACC 11320T=DSM 17175T).

The sphingomonad group contains bacterial isolates that are quite diverse in terms of their phylogenetic, ecological and physiological properties. Thus, the genus Sphingomonas was divided into four distinct genera, Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis on the basis of 16S rRNA gene sequence phylogenetic analysis, signature nucleotides, fatty acid profiles and polyamine patterns and this classification is currently widely accepted. In this study, a complete analysis of the 16S rRNA gene sequences of all the members of the group of sphingomonads encompassed in the genera Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis was inferred by using tree-making algorithms. [Sphingomonas] xenophaga DSM 6383T was found to form a distinct clade with the members of the genus Sphingobium, whereas [Sphingomonas] taejonensis DSM 15583T forms a clade with the members of the genus Sphingopyxis. The respective positions of these strains were also supported by the data for signature nucleotides, 2-hydroxy fatty acid profiles, polyamine patterns and the nitrate reduction properties of the strains. We therefore propose the reclassification of [Sphingomonas] xenophaga and [Sphingomonas] taejonensis as Sphingobium xenophagum comb. nov. (type strain DSM 6383T=CIP 107206T) and Sphingopyxis taejonensis comb. nov. (type strain DSM 15583T=KCTC 2884T=KCCM 41068T), respectively.

A polyphasic taxonomic study of the two subspecies of Paenibacillus larvae, Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens, supported the reclassification of the subspecies into one species, Paenibacillus larvae, without subspecies separation. Our conclusions are based on the analysis of six reference strains of P. larvae subsp. pulvifaciens and three reference strains and 44 field isolates of P. larvae. subsp. larvae. The latter originated from brood or honey of clinically diseased honey bee colonies or from honey of both clinically diseased and asymptomatic colonies from Sweden, Finland and Germany. Colony and spore morphology, as well as the metabolism of mannitol and salicin, did not allow a clear identification of the two subspecies and SDS-PAGE of whole-cell proteins did not support the subspecies differentiation. For genomic fingerprinting, repetitive element-PCR fingerprinting using ERIC primers and PFGE of bacterial DNA were performed. The latter method is a high-resolution DNA fingerprinting method proven to be superior to most other methods for biochemical and molecular typing and has not previously been used to characterize P. larvae. ERIC-PCR identified four different genotypes, while PFGE revealed two main clusters. One cluster included most of the P. larvae subsp. larvae field isolates, as well as all P. larvae subsp. pulvifaciens reference strains. The other cluster comprised the pigmented variants of P. larvae subsp. larvae. 16S rRNA gene sequences were determined for some strains. Finally, exposure bioassays demonstrated that reference strains of P. larvae subsp. pulvifaciens were pathogenic for honey bee larvae, producing symptoms similar to reference strains of P. larvae subsp. larvae. In comparison with the type strain for P. larvae subsp. larvae, ATCC 9545T, the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT100. An emended description of the species is given.

Six strains with more than 99·5 % 16S rRNA gene sequence similarity, identical internal spacer region profiles and restriction analysis of the amplified 16S rRNA gene patterns were isolated from fermenting grape musts during independent studies carried out in France and Spain many years apart. Strains are Gram-positive, motile, facultatively anaerobic rods that do not exhibit catalase activity and have the ability to utilize pentose sugars (ribose and/or l-arabinose), although they are homofermentative bacteria. Strains ferment pentoses exclusively yielding lactic acid as the end product. A broad set of molecular techniques has been applied to characterize these strains and the results show a high degree of genotypical congruence, sharing identical profiles with 16S rRNA-based techniques. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the genus Lactobacillus, closely related to Lactobacillus mali, Lactobacillus nagelii and Lactobacillus satsumensis (with approximately 95 % sequence similarity). DNA–DNA hybridization experiments confirmed the independent status at the species level of these fermenting grape-musts strains. Phenotypically they can be distinguished from the closest relatives by several traits such as growth temperatures and fermentation of carbohydrates. The name Lactobacillus vini sp. nov. is proposed, with strain Mont 4T (=DSM 20605T=CECT 5924T) as the type strain.

Three enterococci constituted two aberrant branches after numerical analysis of (GTG)5-PCR fingerprints: analogous patterns were found for two water isolates, strains W213 and W442T, and a separate position was found for an isolate from the gut of a termite, strain LMG 8895T. 16S rRNA gene sequence analysis classified all three strains in the Enterococcus faecalis species group. Further sequencing analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase α-subunit) and whole-cell-protein analysis confirmed a distinct position for the two water isolates and the termite strain, respectively. DNA–DNA hybridization experiments and distinct phenotypic features between the strains studied and representatives of the E. faecalis species group confirmed novel species status, respectively, for the two water isolates, strains W213 and W442T, and for strain LMG 8895T. The names Enterococcus silesiacus sp. nov. and Enterococcus termitis sp. nov. are proposed for the novel taxa, with W442T (=CCM 7319T=LMG 23085T) and LMG 8895T (=CCM 7300T) as the respective type strains.

A polyphasic taxonomic study of the bacterial flora isolated from traditional French wheat sourdough, using phenotypic characterization and phylogenetic as well as genetic methods, revealed a consistent group of isolates that could not be assigned to any recognized species. These results were confirmed by randomly amplified polymorphic DNA and amplified fragment length polymorphism fingerprinting analyses. Cells were Gram-positive, homofermentative rods. Comparative 16S rRNA gene sequence analysis of the representative strain LP33T indicated that these strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus paralimentarius and Lactobacillus mindensis. DNA–DNA reassociation experiments with the three phylogenetically closest Lactobacillus species confirmed that LP33T (=DSM 16982T=CIP 108546T=TMW 1.1265T) represents the type strain of a novel species, for which the name Lactobacillus nantensis sp. nov. is proposed.

Three Gram-positive, moderately halophilic, spore-forming rods, designated XH-63T, XH-62 and EJ-15, were isolated from two salt lakes located near Xilin Hot and Ejinor, in Inner Mongolia, China. The strains were strictly aerobic and motile, with spherical, terminal and deforming endospores. They grew at pH 5·0–9·0 (optimal growth at pH 7·5), between 4 and 45 °C (optimal growth at 37 °C) and at salinities of 1–20 % (w/v) total salts, growing optimally at 10 % (w/v) salts. They had meso-diaminopimelic acid in the cell wall peptidoglycan and DNA G+C contents of 36·1–37·1 mol%. The polar lipid pattern of strain XH-63T, selected as the representative strain, consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and a phospholipid and two amino phospholipids of unknown structure. This strain possessed anteiso-C15 : 0 and anteiso-C17 : 0 as the major fatty acids (altogether representing 72·5 % of total) and MK-7 as the major menaquinone. 16S rRNA gene analysis of the three strains showed that they were within the Gracilibacillus cluster, with highest sequence similarity (95·4–95·8 %) with Gracilibacillus dipsosauri. Based on a combination of phenotypic, chemotaxonomic and phylogenetic features, it is proposed that the three isolates represent a novel species of the genus Gracilibacillus, Gracilibacillus orientalis sp. nov. The type strain is strain XH-63T (=CCM 7326T=AS 1.4250T=CECT 7097T).

A strictly anaerobic, mesophilic, endospore-forming, lipolytic bacterium, designated strain R1T, was isolated from bovine rumen fluid and characterized. Cells of this isolate were Gram-positive, non-motile rods that formed spherical terminal spores. The overall biochemical and physiological characteristics indicated that this strain should be placed in the genus Clostridium. The strain grew at temperatures between 25 and 47 °C (optimum, 37 °C), at pH between 5·0 and 8·5 (optimum pH 5·5–7·0) and in NaCl concentrations of 0–3 % (w/v). The isolate was not able to utilize glucose or other carbohydrates as carbon sources. The DNA G+C content was 31·2 mol%. Sequence analysis of the 16S rRNA gene of R1T revealed that it has the closest match (98 % similarity) with Clostridium tetanomorphum DSM 4474T. The highest levels of DNA–DNA relatedness of the isolate were 61·9 and 54·3 % with Clostridium pascui DSM 10365T and C. tetanomorphum DSM 4474T, respectively. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G+C content, DNA–DNA hybridization data and distinct phenotypic characteristics, strain R1T (=DSM 17049T=CCUG 50446T) was classified in the genus Clostridium, as a member of a novel species, for which the name Clostridium lundense sp. nov. is proposed.

The taxonomic position of two spore-forming strains 6T19T and 6T29, isolated from cotton composts for the cultivation of oyster mushroom (Pleurotus ostreatus), was investigated by a polyphasic approach. Cells of strains 6T19T and 6T29 were rod-shaped, Gram-negative and strictly aerobic. Sequencing and comparative analyses for the 16S rRNA genes of these strains clearly showed their phylogenetic affiliation to the genus Ureibacillus. Their closest relatives Ureibacillus thermosphaericus and Ureibacillus terrenus have sequence similarity of 96·9 and 97·5 %, respectively. The isoprenoid quinones of isolate 6T19T were MK-9, MK-8, MK-7, MK-10 and MK-6 (45 : 27 : 18 : 5 : 4 %), the peptidoglycan type was l-lys←d-Asp and the main cellular fatty acid was i-C16 : 0. DNA–DNA hybridization experiments resulted in relatedness values of 37 % between 6T19T and U. thermosphaericus DSM 10633T and 41 % between 6T19T and U. terrenus DSM 12654T. Based on the polyphasic data, strains 6T19T and 6T29 can be described as members of a novel species of the genus Ureibacillus, for which the name Ureibacillus suwonenesis sp. nov. is proposed. The type strain is 6T19T (=KACC 11287T=DSM 16752T).