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A novel bacterial strain, TLK-CK17

A novel proteobacterial strain designated SYSU H10001T was isolated from a soil sample collected from plateau meadow in Hongyuan county, Sichuan province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities and phylogenetic analysis, strain SYSU H10001T was most closely related to Lysobacter soli KCTC 22011T (98.6%, sequence similarity) and Lysobacter panacisoli JCM 19212T (98.2%). The prediction result of secondary metabolites based on genome shown that the strain SYSU H10001T contained 3 clusters of bacteriocins, 1 cluster of non-ribosomal peptide synthetase, 1 cluster of type 1 polyketide synthase and 1 cluster of arylpolyene. In addition, the major isoprenoid quinone was Q-8 and the major fatty acids were identified as iso-C15:0, iso-C17:0 and Summed feature 9. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and three unidentified phospholipids. The genomic DNA G + C content of strain SYSU H10001T was 66.5% (genome). On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU H10001T represents a novel species of the genus Lysobacter, for which the name Lysobacter prati sp. nov. is proposed. The type strain is SYSU H10001T (= KCTC 72062T = CGMCC 1.16662T).

A novel bacterial strain, designated KVB24T, was isolated from sea-water of Busan Harbour in South Korea. Cells of strain KVB24T were Gram-stain negative, aerobic, rod shaped and non-motile. Strain KVB24T grew optimally at 25–28 °C and pH 6.5–7.0. Based on 16S rRNA gene sequence analysis, strain KVB24T was shown to belong to the genus Lysobacter within the class Gammaproteobacteria and to be closely related to Lysobacter dokdonensis DS-58T, Lysobacter hankyongensis KTce-2T and Lysobacter niastensis GH41-7T. DNA–DNA relatedness between strain KVB24T and its current closest relative was below 70%. The predominant fatty acids of strain KVB24T were iso-C11:0, iso-C11:0 3-OH, iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0 and summed feature 9 comprising (iso-C17:1 ω9c and/or 10 methyl C16:0); the prominent isoprenoid was Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G + C content of genomic DNA from strain KVB24T was determined to be 67.5 mol%. Based on the phenotypic, genotypic and chemotaxonomic analyses, strain KVB24T represents a novel species of the genus Lysobacter, for which the name Lysobacter caseinilyticus sp. nov. is proposed. The type strain is KVB24T (= KACC19816T = JCM32879T).

Two bacterial strains, designated D10T and U8T, were isolated from soil samples from the Dong-angyeong cave and Geommeolle wharf sea-coast, Udo-Island, Jeju, South Korea. Both novel bacterial strains are yellow-pigmented, Gram-stain negative, motile by means of monotrichous flagella, short rod shaped and strictly aerobic. A phylogenetic tree was reconstructed based on their 16S rRNA gene sequences, which indicated that these two strains belong to the genus Lysobacter within the family Xanthomonadaceae. Strain D10T showed high 16S rRNA gene sequence similarities with Lysobacter humi FJY8T (99.0%), Lysobacter xinjiangensis RCML-52T (98.9%) and Lysobacter mobilis 9NM-14T (97.2%), whereas strain U8T showed high sequence similarities to L. mobilis 9NM-14T (97.9%), L. xinjiangensis RCML-52T (97.8%), L. humi FJY8T (97.5%) and Lysobacter bugurensis ZLD-29T (97.1%). The 16S rRNA gene sequence similarity between D10T and U8T was 97.0%. Strain D10T showed low DNA–DNA relatedness to U8T (57.7 ± 3.4%), L. humi FJY8T (48.8 ± 4.3%), L. xinjiangensis RCML-52T (60.1 ± 2.4%) and L. mobilis 9NM-14T (55.9 ± 1.9%). The level of DNA–DNA relatedness for strain U8T with respect to D10T, L. mobilis 9NM-14T, L. xinjiangensis RCML-52T, L. humi FJY8T, and L. bugurensis ZLD-29T was 55.5 ± 0.5%, 54.5 ± 2.1%, 58.1 ± 0.8%, and 51.9 ± 3.4%, respectively. The major polar lipids for both strains were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids for both strains were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17:1 ω9c/C16:0 10-methyl), and ubiquinone (Q-8) as the only isoprenoid quinone for both strains. The DNA G + C contents of the strains D10T and U8T were determined to be 70.2 mol% and 70.6 mol%. On the basis of phenotypic, genotypic, chemotaxonomic, and phylogenetic analysis, both strains D10T and U8T represent a novel species in the genus Lysobacter, for which the names Lysobacter helvus sp. nov. and Lysobacter xanthus sp. nov. are proposed, respectively. The type strain of L. helvus is D10T (= KCTC 62111T = JCM 32364T) and the type strain of L. xanthus is U8T (= KCTC 62112T = JCM 32365T).

A Gram-stain negative, rod-shaped bacterial, catalase and oxidase positive strain (83-4T) that formed yellow colonies was isolated from human Meibomian gland secretions. Strain 83-4T belongs to the genus Lysobacter according to phylogenetic analysis based on 16S rRNA gene sequences. The DNA G+C content was 67.1 mol%. The circular genome was 2.6 Mb, which contained 2431 protein-coding sequences, 75 pseudogenes, 46 tRNAs, 3 rRNAs and 4 ncRNAs. A bacteriocin cluster and aryl polyene cluster were also found in the genome. The average nucleotide identity value was 79.6% between isolate 83-4T and the closely related type strain Lysobacter tolerans UM1T. The estimated DNA–DNA hybridization value between strain 83-4T and L. tolerans UM1T was 41.6%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Iso-C15:0, iso-C11:0 3-OH, iso-C11:0 and summed feature 9 (iso-C17:1ω9c) were the major fatty acids. Ubiquinone (Q-8) was the only respiratory quinone. Therefore, based on the data of phylogenetic analysis, chemotaxonomical and biochemical analyses, it is concluded that strain 83-4T represents a novel species of the genus Lysobacter with the name of Lysobacter oculi sp. nov. The type strain is 83-4T (= CGMCC 1.13464T = NRBC 113451T).

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.

A Gram-staining negative, aerobic, rod-shaped bacterium, designated YIM C01544(T), was isolated from a soil sample collected from Sigangli Cave, Yunnan province, South-West China. The strain was able to grow over a range of temperatures (4-30 °C), pH (6.0-10.0) and NaCl concentration (0-2 %, w/v). Comparative 16S rRNA gene sequence analysis revealed that strain YIM C01544(T) should be a member of the genus Lysobacter. The strain is closely related to Lysobacter niastensis GH41-7(T) (97.6 %), Lysobacter soli DCY21(T) (97.5 %), Lysobacter enzymogenes DSM 2043(T) (97.3 %), Lysobacter antibioticus DSM 2044(T) (97.1 %) and Lysobacter panacisoli CJ29(T) (97.1 %). The genomic DNA relatedness values (

A Gram-negative and aerobic bacterium, designated YIM 77875(T), was isolated from a geothermal soil sample collected at Rehai National Park, Tengchong, Yunnan Province, south-west China. Bacterial growth occurred from 37 to 65 °C (optimum 50 °C), pH 6.0-8.0 (optimum pH 7.0) and 0-1 % NaCl (w/v). Cells were rod-shaped and colonies were convex, circular, smooth, yellow and non-transparent. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM 77875(T) belongs to the genus Lysobacter. The 16S rRNA gene sequence similarity values between strain YIM 77875(T) and other species of the genus Lysobacter were all below 94.7 %. The polar lipids of strain YIM 77875(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and five unknown phospholipids. The predominant respiratory quinone was Q-8 and the G+C content was 68.8 mol%. Major fatty acids were iso-C(16:0), iso-C(15:0) and iso-C(11:0). On the basis of the morphological and chemotaxonomic characteristics, as well as genotypic data, strain YIM 77875(T) represents a novel species, Lysobacter thermophilus sp. nov., in the genus Lysobacter. The type strain is YIM 77875(T) (CCTCC AB 2012064(T) = KCTC 32020(T)).

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells. Electronic supplementary material The online version of this article (doi:10.1007/s10482-012-9746-7) contains supplementary material, which is available to authorized users.

A bacterial strain, designated as THG-SKA3(T), was isolated from field soil of Kyung Hee University, South Korea. Cells of the isolate were observed to be Gram-negative, aerobic, rod-shaped and motile by gliding. The strain was found to grow optimally at 28 °C, at pH 7 and in absence of NaCl. Based on 16S rRNA gene sequence comparisons, strain THG-SKA3(T) shared highest sequence similarity with Lysobacter niastensis KACC 11588(T) followed by Lysobacter panacisoli KACC 17502(T), Lysobacter enzymogenes LMG 8762(T) and Lysobacter oryzae KCTC 22249(T). The G+C content of THG-SKA3(T) was determined to be 68.9 mol%. The DNA-DNA relatedness values between strain THG-SKA3(T) and its closest phylogenetic neighbors were below 25.0 %.The major polar lipids of strain THG-SKA3(T) were determined to be diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant respiratory quinone was identified as ubiquinone 8 (Q-8). The major cellular fatty acids were identified as branched chain iso-C15:0, iso-C16:0 and unsaturated iso-C17:1 ω9c. On the basis of polyphasic data presented, it is evident that strain THG-SKA3(T) represents a novel species of the genus Lysobacter, for which the name Lysobacter agri sp. nov. (type strain THG-SKA3(T) = KACC 18283(T) = CSCTCC AB 2015126(T)) is proposed.

Bacterial diversity in the rhizosphere of beach morning glory (Calystegia soldanella) and wild rye (Elymus mollis), two of the major plant species inhabiting the coastal sane dune in Tae-An, Korea, was studied by the analysis of community 16S rRNA gene clones. The amplified rDNA restriction analysis (ARDRA) of the clones using HaeIII exhibited significant differences in the community composition between the two plant species as well as regional differences, but also identified a specific ARDRA pattern that was most common among the clones regardless of plant species. Subsequent sequence analysis indicated that the pattern was that of Lysobacter spp., which is a member of the family Xanthomonadaceae, class Gamma proteobacteria. The Lysobacter clones comprised 50.6% of the clones derived from C. soldanella and 62.5% of those from E. mollis. Other minor patterns included those of Pseudomonas spp., species of Rhizobium, Chryseobacterium spp. and Pantoea spp. among C. soldanella clones, and Pseudomonas sp. and Aeromonas hydrophila among E. mollis clones. It is not yet clear what kind of roles Lysobacter plays in association with sand dune plants, but its universal presence in the rhizosphere, together with the potential of this taxon for antagonistic activity against plant pathogens, suggests that Lysobacter might form a symbiotic relationship with its host plants.

Two bacterial strains, designated THG-DN8.7(T) and THG-DN8.3(T), were isolated from the rhizosphere of a strawberry plant in Gyeryong Mountain, South Korea. Cells of both isolates were observed to be Gram-negative, yellow-coloured and rod-shaped. Comparative 16S rRNA gene sequence analysis showed that strain THG-DN8.7(T) had highest sequence similarities to Lysobacter yangpyeongensis KACC 11407(T) (97.2 %), Lysobacter niabensis KACC 11587(T) (97.0 %) and Lysobacter oryzae KCTC 22249(T) (96.9 %), while strain THG-DN8.3(T) had closely similarity with L. niabensis KACC 11587(T) (98.1 %), L. oryzae KCTC 22249(T) (97.1 %) and L. yangpyeongensis KACC 11407(T) (96.1 %). DNA-DNA relatedness values between strains THG-DN8.7(T) and THG-DN8.3(T) and their closest phylogenetically neighbours were below 30.0 %, which indicates that strains THG-DN8.7(T) and THG-DN8.3(T) represent distinct species within the genus Lysobacter. Both strains were found to contain iso-C15:0, iso-C16:0 and iso-C17:1 ω9c as predominant fatty acids and ubiquinone-8 as major isoprenoid quinone. The major polar lipids were identified as phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of strains THG-DN8.7(T) and THG-DN8.3(T) were determined to be 66.9 and 67.8 mol%, respectively. These data are consistent with the affiliation of the two new species represented by THG-DN8.7(T) and THG-DN8.3(T) to the genus Lysobacter. The names Lysobacter fragariae sp. nov. and Lysobacter rhizosphaerae sp. nov. are proposed for these species with the type strains THG-DN8.7(T) (=KCTC 42236(T) = JCM 30322(T)) and THG-DN8.3(T) (=KCTC 42237(T) = JCM 30321(T)), respectively.

A Gram-stain negative, aerobic, rod-shaped and non-spore-forming bacterium, CC-Bw-6(T) was isolated from chopped tomato stems. Analysis of the 16S rRNA gene sequence of the strain CC-Bw-6(T) showed its affiliation with the genus Lysobacter within the class Gammaproteobacteria. Strain CC-Bw-6(T) was found to be most closely related to Lysobacter panaciterrae KCTC 12601(T) (97.0%) and Lysobacter daecheongensis KCTC 12600(T) (96.8%), and showed lower similarity (