Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

<Emphasis Type="Italic">Virgibacillus litoralis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from saline soil

A Gram-positive, moderately halophilic, endospore-forming, catalase- and oxidase-positive, motile, rod-shaped, aerobic bacterium, designated strain JSM 089168^T, was isolated from saline soil collected from Naozhou Island, Leizhou Bay, South China Sea. The organism was able to grow with 2–25% (w/v) total salts (optimum, 5–10%), at pH 6.0–10.0 (optimum, pH 8.0) and 10–45°C (optimum, 30°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The strain contained MK-7 as the predominant menaquinone, and diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids. The major cellular fatty acids were anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0, and the DNA G + C content was 40.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 089168^T should be assigned to the genus Virgibacillus, being related most closely to the type strains of Virgibacillus carmonensis (sequence similarity 97.6%), Virgibacillus necropolis (97.3%) and Virgibacillus halodenitrificans (97.1%). Levels of DNA–DNA relatedness between strain JSM 089168^T and the type strains of V. carmonensis, V. necropolis and V. halodenitrificans were 20.4, 14.3 and 12.0%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 089168^T represents a novel species of the genus Virgibacillus, for which the name Virgibacillus litoralis sp. nov. is proposed. The type strain is JSM 089168^T (=DSM 21085^T =KCTC 13228^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 089168^T is FJ425909.     

<Emphasis Type="Italic">Arthrobacter halodurans</Emphasis> sp. nov., a new halotolerant bacterium isolated from sea water

A novel Gram-positive, halotolerant, non-sporulating, non-motile, catalase-positive, oxidase-negative and aerobic bacterium, designated strain JSM 078085^T, was isolated from sea water collected from the South China Sea. Strain JSM 078085^T exhibited a rod-coccus growth cycle and produced a yellow pigment. The strain was able to grow in the presence of 0–12% (w/v) NaCl and at pH 6.0–9.5 and 4–35°C; optimum growth was observed at pH 7.0 and 25–30°C in the absence of NaCl. The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu). Cell-wall sugars contained galactose and glucose. Strain JSM 078085^T contained menaquinone MK-9(H₂) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The major cellular fatty acids were anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0 and the DNA G + C content was 63.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 078085^T should be assigned to the genus Arthrobacter, being most closely related to the type strain of Arthrobacter rhombi (sequence similarity 97.1%), and the two strains formed a distinct lineage in the phylogenetic tree. The level of DNA–DNA relatedness between strain JSM 078085^T and the type strain of Arthrobacter rhombi was 10.6%. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078085^T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter halodurans sp. nov. is proposed. The type strain is JSM 078085^T (=DSM 21081^T=KCTC 19430^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078085^T is EU583729.     

<Emphasis Type="Italic">Halomonas qijiaojingensis</Emphasis> sp. nov. and <Emphasis Type="Italic">Halomonas flava</Emphasis> sp. nov., two moderately halophilic bacteria isolated from a salt lake

Two moderately halophilic, Gram-negative, rod-shaped bacteria, designated YIM 93003^T and YIM 94343^T, were isolated from a salt lake in Xinjiang province, north-west China. The two strains YIM 93003^T and YIM 94343^T grew at 20–40°C, pH 6–9, 0.5–24% (w/v) NaCl and at 20–40°C, pH 6–9, 0.5–23% (w/v) NaCl, respectively. No growth occurred in absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 93003^T and YIM 94343^T were phylogenetically affiliated to the genus Halomonas and exhibited sequence similarity of 97.5% and 97.4% to the type strain Halomonas anticariensis DSM 16096^T, respectively. The strains possessed chemotaxonomic markers that were consistent with their classification in the genus Halomonas (Q-9 as predominant respiratory quinine; C18:1ω7c, C16:0 and C16:1 ω7c/iso-C15:02-OH as the major fatty acids). The DNA–DNA hybridization values for strains YIM 93003^T and YIM 94343^T, YIM 93003^T and DSM 16096^T, YIM 94343^T and DSM 16096^T were 38.1 ± 3.0, 18.3 ± 4.7, and 20.8 ± 4.6%, respectively. The G+C contents of the strains YIM 93003^T and YIM 94343^T were 63.4 and 64.0 mol%, respectively. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA–DNA hybridization results, two novel species, Halomonas qijiaojingensis sp. nov., and Halomonas flava sp. nov., are proposed. The type strains are YIM 93003^T (=CCTCC AB 208133^T =KCTC 22228^T) and YIM 94343^T (=CCTCC AB 2010382^T =KCTC 23356^T), respectively.     

<Emphasis Type="Italic">Halobacillus naozhouensis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from a sea anemone

A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071068^T, was isolated from a sea anemone (Anthopleura xanthogrammica) collected from the Naozhou Island on the Leizhou Bay in the South China Sea. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071068^T was able to grow with 1–20% (w/v) total salts (optimum, 6–9%), at pH values of 6.0–10.0 (optimum, pH 7.5) and a temperature range of 10–35°C (optimum, 25°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and iso-C_15:0. The genomic DNA G + C content was 42.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 071068^T belonged to the genus Halobacillus. The 16S rRNA gene sequence similarities between strain JSM 071068^T and the type strains of the recognized Halobacillus species ranged from 97.9% (with Halobacillus alkaliphilus) to 95.3% (with Halobacillus kuroshimensis). The levels of DNA–DNA relatedness between the new isolate and the type strains of H. alkaliphilus, Halobacillus campisalis, Halobacillus halophilus and Halobacillus seohaensis were 25.6, 22.1, 10.8 and 13.2%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 071068^T represents a new species of the genus Halobacillus, for which the name Halobacillus naozhouensis sp. nov. is proposed, with JSM 071068^T (=DSM 21183^T =KCTC 13234^T) as the type strain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 071068^T is EU925615.     

<Emphasis Type="Italic">Tindallia texcoconensis</Emphasis> sp. nov., a new haloalkaliphilic bacterium isolated from lake Texcoco,Mexico

A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300^T) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300^T was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35°C with absence of growth above 45°C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300^T has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA–DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300^T is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300^T = DSM 18041^T = JCM 13990^T).     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

<Emphasis Type="Italic">Pseudonocardia serianimatus</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized leaves of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

An aerobic, non-motile, catalase-positive, Gram-stain positive actinomycete designated YIM 63233^T was isolated from the surface-sterilized leaves of Artemisia annua L. and characterized using a polyphasic taxonomic approach. Optimal growth occurred at 20–28°C, pH 6.0–7.0 and in the presence of 0–3% (w/v) NaCl. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 63233^T clustered with species of the genus Pseudonocardia, displaying ≥1.2% sequence divergence with recognized species of this genus (from 98.8 to 94.0%). Relatively low levels of DNA–DNA relatedness were found between strain YIM 63233^T and Pseudonocardia petroleophila IMSNU 22072^T, which supported the classification of strain YIM 63233^T within a novel species of the genus Pseudonocardia. The G + C content of genomic DNA was 72.0 mol%. Strain YIM 63233^T possessed chemotaxonomic markers that were consistent with classification in the genus Pseudonocardia, i.e. the predominant fatty acids were iso-C16:0 (32.27%), C16:0 10-methyl (8.73%) and C17:1ω8c (8.30%), whilst the predominant menaquinone was MK-8(H₄). The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. The major cell wall sugars were glucose, arabinose, galactose, mannose and rhamnose. The results of physiological and biochemical tests and DNA–DNA hybridization allowed the phenotypic and genotypic differentiation of strain YIM 63233^T from its closest phylogenetic neighbours. Therefore, the new isolate YIM 63233^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia serianimatus sp. nov. is proposed. The type strain is YIM 63233^T (=DSM 45302^T = CCTCC AA 208079^T).     

Role of trehalose synthesis pathways in salt tolerance mechanism of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> f. sp. <Emphasis Type="Italic">denitrificans</Emphasis> IL106

Two strains of trichloroethylene (TCE)-degrading bacteria were isolated from soils at polluted and unpolluted sites. The isolates, strains TE26^T and K6, showed co-substrate-independent TCE-degrading activity. TCE degradation was accelerated by preincubation with tetrachloroethylene, cis-dichloroethylene (DCE) and 1,1-DCE. TCE-degrading activities of strains TE26^T and K6 were 0.23, 0.24 mol min^–1 g^–1 dry cells, respectively. 16S rDNA sequences of strains TE26^T and K6 were almost identical (99.7% similarity), and most closely related to Ralstonia basilensis (ATCC17697^T) (98.5% similarity). From the results of DNA–DNA hybridizations, strain TE26^T was genetically coherent to strain K6 (94 and 88% hybridization), and exhibited lower relatedness to R. basilensis (DSM11853^T) (44% and 15%). In addition, because of the differences in chemotaxonomic properties, strain TE26^T and strain K6 appear to be distinct from all established species of the Ralstonia group. Based on these results and the proposal of transferring R. basilensis and related species to Wautersia gen. nov., we propose that these strains should be assigned to the genus Wautersia as Wautersia numadzuensis sp. nov.     

<Emphasis Type="Italic">Halomonas sediminis</Emphasis> sp. nov., a new halophilic bacterium isolated from salt-lake sediment in China

A novel Gram-negative, slightly halophilic, catalase- and oxidase-positive, obligately aerobic bacterium, strain YIM-C248^T, was isolated from a sediment sample collected from a salt-lake in the Qaidam Basin in Qinghai, north-west China. Cells were non-sporulating short rods, occurring singly or as doublets, motile with peritrichous flagella. Growth occurred with 1–15% (w/v) NaCl [optimum 2–4% (w/v) NaCl], at pH 6.0–10.0 (optimum pH 7.5) and at 4–35°C (optimum 25–30°C). The major cellular fatty acids were C_18:1 ω7c, C_12:0 3-OH, cyclo C_19:0 ω8c, C_16:0 and C_16:1. The predominant respiratory quinone was Q-9 and the genomic DNA G + C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM-C248^T should be assigned to the genus Halomonas. The sequence similarities between the isolate and the type strains of members of the genus Halomonas were in the range of 92.5–97.5%. The combination of phylogenetic analysis, DNA–DNA hybridization data, phenotypic characteristics and chemotaxonomic differences supported the view that strain YIM-C248^T represents a new species of the genus Halomonas, for which the name Halomonas sediminis sp. nov. is proposed, with YIM-C248^T (=CCTCC AA 207031 = KCTC 22167) as the type strain. The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence of strain YIM-C248^T is EU135707.     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

<Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> subsp. <Emphasis Type="Italic">kleinii</Emphasis> subsp. nov. (Enterobacteriales: Enterobacteriaceae)

Association between bacteria Photorhabdus and their nematode hosts Heterorhabditis represents one of the emerging models in symbiosis studies. In this study, we isolated the bacterial symbionts of the nematode Heterorhabditis georgiana. Using gyrB sequences for phylogenetic analysis, these strains were shown to be part of the species of Photorhbdus luminescens but with clear separation from currently recognized subspecies. Physiological properties and DNA–DNA hybridization profiles also supported the phylogenetic relationship of these strains. Therefore, a new subspecies, Photorhabdus luminescens subsp. kleinii subsp. nov., is proposed with the type strain KMD37^T (=DSM 23513 =ATCC =NRRL B-59419).     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

<Emphasis Type="Italic">Marinobacter zhanjiangensis</Emphasis> sp. nov., a marine bacterium isolated from sea water of a tidal flat of the South China Sea

A novel Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, aerobic bacterium, designated strain JSM 078120^T, was isolated from sea water collected from a tidal flat of Naozhou Island, South China Sea. Growth occurred with 1–15% (w/v) total salts (optimum, 2–4%), at pH 6.0–10.0 (optimum, pH 7.5) and at 4–35°C (optimum, 25–30°C). The major cellular fatty acids were C_18:1 ω9c, C_16:0, C_12:0 3-OH and C_16:1 ω7c. The predominant respiratory quinone was ubiquinone Q-9, and the genomic DNA G + C content was 60.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 078120^T should be assigned to the genus Marinobacter, being related most closely to the type strains of Marinobacter segnicrescens (sequence similarity 98.2%), Marinobacter bryozoorum (97.9%) and Marinobacter gudaonensis (97.6%). The sequence similarities between the novel isolate and the type strains of other recognized Marinobacter species ranged from 96.7 (with Marinobacter salsuginis) to 93.3% (with Marinobacter litoralis). The levels of DNA–DNA relatedness between strain JSM 078120^T and the type strains of M. segnicrescens, M. bryozoorum and M. gudaonensis were 25.3, 20.6 and 18.8%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078120^T represents a novel species of the genus Marinobacter, for which the name Marinobacter zhanjiangensis sp. nov. is proposed. The type strain is JSM 078120^T (= CCTCC AB 208029^T = DSM 21077^T = KCTC 22280^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078120^T is FJ425903.     

The use of <Emphasis Type="Italic">gyrB</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1.