- Volume 60, Issue 12, 2010

Five Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped bacterial isolates were obtained from infant formula and an infant formula production environment and were investigated by use of a polyphasic taxonomic study. Biochemical tests and partial rpoB gene sequence analysis of the five isolates revealed that they formed two distinct groups in the family Enterobacteriaceae, closely related to several species of the genera Pantoea and Erwinia, which indicated a phylogenetic position within the genus Pantoea or the genus Erwinia. Multilocus sequence analysis of concatenated partial atpD, gyrB, infB and rpoB gene sequences of two of the isolates suggested that they represented two novel species of the genus Pantoea, phylogenetically related most closely to Pantoea septica. The five isolates had general characteristics consistent with those of the genus Pantoea, and DNA–DNA hybridizations between two representatives and the type strains of their phylogenetically closest relatives based on comparative 16S rRNA gene sequence analysis showed that the isolates represented two novel genospecies. These two genospecies could be differentiated from each other based on fermentation of galacturonate, sorbitol and potassium 5-ketogluconate. They could be differentiated from phylogenetically related Pantoea species based on their ability to ferment lactose and to utilize β-gentiobiose and raffinose, their inability to ferment or utilize d-arabitol, and their inability to produce indole. On the basis of the results obtained, the five isolates are considered to represent two novel species of the genus Pantoea, for which the names Pantoea gaviniae sp. nov. (type strain A18/07T =LMG 25382T =DSM 22758T) and Pantoea calida sp. nov. (type strain 1400/07T =LMG 25383T =DSM 22759T) are proposed.

Two Gram-negative, non-motile, non-pigmented and curved rod-shaped bacterial strains, designated IMCC4489T and IMCC4451, were isolated from a tidal flat sediment of the Yellow Sea. Strains IMCC4489T and IMCC4451 shared 99.9 % 16S rRNA gene sequence similarity and 78.5 % DNA–DNA relatedness, which suggested that they belonged to the same species. The isolates were most closely related to Reinekea blandensis MED297T (98.7–98.8 % 16S rRNA gene sequence similarity) and Reinekea marinisedimentorum DSM 15388T (95.3–95.4 %). DNA–DNA relatedness between the strains and R. blandensis CCUG 52066T was 31–34 %. Strains IMCC4489T and IMCC4451 could also be differentiated from the type strains of the two recognized Reinekea species by several phenotypic properties. The DNA G+C content was 51.3–51.5 mol% and the major isoprenoid quinone was Q-8. On the basis of the data obtained in this study, it is proposed that strains IMCC4489T and IMCC4451 represent a novel species, Reinekea aestuarii sp. nov. The type strain is IMCC4489T (=KCTC 22813T =KCCM 42938T =NBRC 106079T).

A novel nitrogen-fixing strain, designated BV-ST, was isolated from a sulfur bacterial mat collected from a sulfide spring of the Stavropol Krai, North Caucasus, Russia. Strain BV-ST grew optimally at pH 7.5 and 37 °C. According to the results of phylogenetic analysis, strain BV-ST belonged to the genus Azospirillum within the family Rhodospirillaceae of the class Alphaproteobacteria. Within the genus Azospirillum, strain BV-ST was most closely related to Azospirillum doebereinerae GSF71T, A. picis IMMIB TAR-3T and A. lipoferum ATCC 29707T (97.7, 97.7 and 97.4 % 16S rRNA gene sequence similarity, respectively). DNA–DNA relatedness between strain BV-ST and A. doebereinerae DSM 13131T, A. picis DSM 19922T and A. lipoferum ATCC 29707T was 38, 55 and 42 %, respectively. Similarities between nifH sequences of strain BV-ST and members of the genus Azospirillum ranged from 94.5 to 96.8 %. Chemotaxonomic characteristics (quinone Q-10, major fatty acid C18 : 1 ω7c and G+C content 67 mol%) were similar to those of members of the genus Azospirillum. In contrast to known Azospirillum species, strain BV-ST was capable of mixotrophic growth under microaerobic conditions with simultaneous utilization of organic substrates and thiosulfate as electron donors for energy conservation. Oxidation of sulfide was accompanied by deposits of sulfur globules within the cells. Based on these observations, strain BV-ST is considered as a representative of a novel species of the genus Azospirillum, for which the name Azospirillum thiophilum sp. nov. is proposed. The type strain is BV-ST (=DSM 21654T =VKM B-2513T).

Four novel isolates (R-40508T, R-40507, R-40903 and R-21419) were obtained from different cnidarian species (Phyllogorgia dilatata, Merulina ampliata and Palythoa caribaeorum) from different places in Brazil and Australia. The novel isolates formed a tight phylogenetic group based on 16S rRNA, recA, topA, ftsZ, mreB and rpoA gene sequences. Their closest phylogenetic neighbours were the type strains of Photobacterium leiognathi, P. rosenbergii and P. halotolerans, sharing 97.1–97.5 % 16S rRNA gene sequence similarity. DNA–DNA hybridization between a representative strain (R-40508T) and the type strains of these Photobacterium species revealed less than 20 % relatedness, showing that the new isolates belong to a novel species. Several phenotypic features allow the differentiation of the novel species from its closest phylogenetic neighbours. It has gelatinase and lipase activity and can utilize melibiose, but it cannot grow on 6 % NaCl. In addition, the novel species has the fatty acid iso-C16 : 0, but lacks the fatty acids C17 : 0, C17 : 0 cyclo, iso-C17 : 0, C17 : 1 ω8c and iso-C17 : 1 ω9c. The name Photobacterium jeanii sp. nov. is proposed for this species, with the type strain R-40508T (=LMG 25436T =CAIM 1817T). The G+C content of the type strain is 45.5 mol%.

A Gram reaction-negative, weakly motile, non-spore-forming, rod-shaped, aerobic bacterium designated strain JLT832T was isolated from surface water of the central Pacific Ocean and formed yellow colonies on rich organic (RO) medium. The strain was oxidase-negative and catalase-positive. Acid was produced from mannitol, glucose, sucrose, lactose, sorbitol, maltose, (+)-trehalose and d-fructose. No acid was produced from d-(+)-xylose. The major cellular fatty acids of strain JLT832T were C18 : 1 ω7c, C14 : 0 2-OH and C16 : 0. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Ubiquinone-10 and spermidine were present as the major quinone and polyamine, respectively. The genomic DNA G+C content of strain JLT832T was 66.0±0.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate formed a tight branch within the family Sphingomonadaceae but was clearly separate from established genera in this family. The sequence similarities between the new isolate and type strains of established genera ranged from 90.5 to 94.9 %. Based on these data, strain JLT832T constitutes a novel genus and species, for which the name Stakelama pacifica gen. nov., sp. nov. is proposed. The type strain of Stakelama pacifica is JLT832T (=CGMCC 1.7294T =LMG 24686T).

A novel facultatively anaerobic, sulfur-oxidizing bacterium, strain skB26T, was isolated from anoxic water of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-negative. Strain skB26T oxidized elemental sulfur and thiosulfate to sulfate as sole energy sources. Strain skB26T was microaerobic and could also utilize nitrate as an electron acceptor, reducing it to nitrogen. Growth was observed at temperatures below 28 °C; optimum growth was observed at 22 °C. The pH range for growth was 6.0–9.0, and the optimum pH was 7.5–8.0. Optimum growth of the isolate was observed in medium without NaCl, and no growth was observed in medium containing more than 220 mM NaCl. The G+C content of genomic DNA was around 59 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the strain was a member of the class Betaproteobacteria, and the closest cultivated relative was ‘Thiobacillus plumbophilus’ DSM 6690, with 93 % sequence similarity. Phylogenetic analyses were also performed using sequences of genes involved in sulfur oxidation, inorganic carbon fixation and nitrate respiration. On the basis of its phylogenetic and phenotypic properties, strain skB26T (=NBRC 105220T =DSM 22764T) is proposed as the type strain of a novel species of a new genus, Sulfuricella denitrificans gen. nov., sp. nov.

A moderately halophilic Gram-staining-negative bacterium, designated strain Y26T, was isolated from a tidal flat of Taean coast in South Korea. Cells were strictly aerobic, motile cocci with a single flagellum and showed catalase- and oxidase-positive reactions. Growth of strain Y26T was observed at 15–35 °C (optimum 25–30 °C), pH 6.0–8.0 (optimum pH 6.5–7.5) and with 1.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %). The predominant fatty acids were C18 : 1 ω7c (66.2 %), C16 : 0 (12.4 %) and C10 : 0 3-OH (5.0 %) and the G+C content of the genomic DNA was 61.0 mol%. Strain Y26T contained ubiquinone-10 (Q-10) as the major respiratory quinone. Comparative 16S rRNA gene sequence analysis showed that strain Y26T formed a distinct phyletic lineage from other genera within the Roseobacter clade of the class Alphaproteobacteria and was most closely related to members of the genera Maribius, Maritimibacter and Palleronia with 93.8–94.6 % sequence similarity. On the basis of chemotaxonomic data and molecular properties, strain Y26T represents a novel genus, Hwanghaeicola, within the family Rhodobacteraceae, for which the name Hwanghaeicola aestuarii gen. nov., sp. nov. is proposed. The type strain is Y26T (=KACC 13705T =DSM 22009T).

A Gram-stain-negative, non-motile, non-spore-forming and short rod- or rod-shaped bacterial strain, designated 22-5T, was isolated from a bluespotted cornetfish, Fistularia commersonii, and subjected to taxonomic study. Strain 22-5T grew optimally at 30 °C and in the presence of 2–5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 22-5T belonged to the genus Paracoccus and joined the cluster comprising Paracoccus homiensis DD-R11T and Paracoccus zeaxanthinifaciens ATCC 21588T, with which strain 22-5T exhibited 97.4 and 96.9 % 16S rRNA gene sequence similarity, respectively. Strain 22-5T exhibited 94.0–96.6 % 16S rRNA gene sequence similarity with the other type strains of species of the genus Paracoccus. Strain 22-5T contained Q-10 as the predominant menaquinone and C18 : 1 ω7c as the predominant fatty acid. In this study, P. zeaxanthinifaciens KCTC 22688T also contained Q-10 as the predominant isoprenoid quinone. The DNA G+C content of strain 22-5T was 63.6 mol%. Strain 22-5T exhibited 44 and 32 % DNA–DNA relatedness to P. homiensis KACC 11518T and P. zeaxanthinifaciens KCTC 22688T, respectively. On the basis of phenotypic, phylogenetic and genetic data, strain 22-5T is considered to represent a novel species of the genus Paracoccus, for which the name Paracoccus fistulariae sp. nov. is proposed. The type strain is 22-5T (=KCTC 22803T =CCUG 58401T).

Strain DCY45T was isolated from soil of a ginseng field in Pocheon Province, Korea. Strain DCY45T was Gram-negative, oxidase- and catalase-positive, motile and rod-shaped and produced yellow pigments on R2A agar. The organism grew optimally at 30 °C and at pH 7.0. The G+C content of the genomic DNA was 65.4 mol%. The predominant respiratory quinone was Q-8. The major fatty acids were iso-C17 : 1 ω9c, iso-C16 : 0 and iso-C15 : 0. Phylogenetic analysis based on the 16S rRNA gene sequence was used to determine the taxonomic position of strain DCY45T, which is most closely related to species of the genus Rhodanobacter, with similarity levels of 96.0–98.4 %; DNA–DNA relatedness with related strains was lower than 60 %. Strain DCY45T differed significantly from related type strains in phenotypic characteristics. On the basis of these phenotypic, genotypic and chemotaxonomic studies, strain DCY45T represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter soli sp. nov. is proposed. The type strain is DCY45T (=KCTC 22620T =JCM 16126T).

A chemo-organotrophic, aerobic, non-motile strain, MWH-BRAZ-DAM2DT, isolated from a freshwater pond in Brazil, was characterized phenotypically, phylogenetically and chemotaxonomically. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain with the genus Limnohabitans (Comamonadaceae, Betaproteobacteria). 16S rRNA gene sequence similarities between the isolate and Limnohabitans curvus MWH-C5T, representing the type species of the genus, and the type strains of Limnohabitans parvus and Limnohabitans planktonicus were 98.2, 96.5 and 97.0 %, respectively. DNA–DNA reassociation analyses with DNA of the type strains of all three previously described Limnohabitans species revealed similarity values in the range 26.2–44.6 %. The predominant fatty acids of the isolate were C16 : 1 ω7c/ω6c, C16 : 0, C12 : 0 and C8 : 0 3-OH, the major quinone was ubiquinone Q-8 and the DNA G+C content was 55.8 mol%. The isolate could be discriminated from the type strains of the three Limnohabitans species by several phenotypic traits including differences in the utilization of several carbon sources. Based on the phylogeny of the isolate and its differences from the three most closely related species, the isolate represents a novel species for which the name Limnohabitans australis sp. nov. is proposed. The type strain is MWH-BRAZ-DAM2DT (=DSM 21646T=CCUG 56719T).

Species of the genus Prosthecomicrobium are noted for their numerous cellular appendages or prosthecae that extend from the cells. This investigation confirms that the genus is polyphyletic based on an extensive analysis of the 16S rRNA gene sequences of several named species of the genus. The analyses indicate that some Prosthecomicrobium species are more closely related to non-prosthecate genera, including Devosia, Labrenzia, Blastochloris, Methylosinus, Mesorhizobium and Kaistia, than they are to other species of the genus Prosthecomicrobium. For this reason, two of the Prosthecomicrobium clades which are polyphyletic with the type species, Prosthecomicrobium pneumaticum, are renamed as new genera. The currently named species Prosthecomicrobium enhydrum, Prosthecomicrobium mishustinii, Prosthecomicrobium consociatum and Prosthecomicrobium litoralum are reclassified in two new genera, Vasilyevaea gen. nov. and Bauldia gen. nov. with four new combinations: Vasilyevaea enhydra comb. nov. (the type species) and Vasilyevaea mishustinii comb. nov., and Bauldia consociata comb. nov. and Bauldia litoralis comb. nov. (the type species). The type strain of Vasilyevaea enhydra is strain 9bT (=ATCC 23634T =VKM B-1376T). The type strain of the other species in this genus is Vasilyevaea mishustinii strain 17T (=VKM B-2499T =CCM 7569T). The type strain of Bauldia litoralis is strain 524-16T (= NCIB 2233T =ATCC 35022T). The type strain of the other species in this genus is Bauldia consociata strain 11T (=VKM B-2498T =CCM 7594T).

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

hsp60 gene sequences were determined for members of the genus Bacteroides and sequence similarities were compared with those obtained for the 16S rRNA gene. Among the 29 Bacteroides type strains, the mean sequence similarity of the hsp60 gene (84.5 %) was significantly less than that of the 16S rRNA gene (90.7 %), indicating a high discriminatory power of the hsp60 gene. Species of the genus Bacteroides were differentiated well by hsp60 gene sequence analysis, except for Bacteroides pyogenes JCM 6294T, Bacteroides suis JCM 6292T and Bacteroides tectus JCM 10003T. The hsp60 gene sequence analysis and the levels of DNA–DNA relatedness observed demonstrated that these three type strains are a single species. Consequently, B. suis and B. tectus are heterotypic synonyms of B. pyogenes. This study suggests that the hsp60 gene is an alternative phylogenetic marker for the classification of species of the genus Bacteroides.