Croceicoccus ponticola sp. nov., a lipolytic bacterium isolated from seawater.

A lipolytic, Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated GM-16T, was isolated from seawater around Pohang in the Republic of Korea. Strain GM-16T grew optimally at 30 °C and in the presence of 1.0-2.0 % (w/v) NaCl. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain GM-16T fell within the clade comprising the type strains of Croceicoccus species. Strain GM-16T exhibited the highest 16S rRNA gene sequence similarity (97.7 %) to the type strain of Croceicoccus pelagius and sequence similarities of 96.3-96.7 % to the type strains of the other Croceicoccus species. Strain GM-16T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) as major fatty acids. The major polar lipids of strain GM-16T were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid and one unidentified glycolipid. The DNA G+C content of strain GM-16T was 62.6 mol%. The mean DNA-DNA relatedness value between strain GM-16T and C. pelagius DSM 101479T was 16 %. The average nucleotide identity values between strain GM-16T and the type strains of C.roceicoccus pelagius, C.roceicoccus marinus, C.roceicoccus naphthovorans and C.roceicoccus mobilis were 83.96-84.44 %. The phylogenetic and genetic data and differential phenotypic properties indicated that strain GM-16T is separated from recognized Croceicoccus species. On the basis of the data presented here, strain GM-16T is considered to represent a novel species of the genus Croceicoccus, for which the name Croceicoccus ponticola sp. nov. is proposed. The type strain is GM-16T (=KACC 19611T=KCTC 62423T=NBRC 113192T).

Altererythrobacter insulae sp. nov., a lipolytic bacterium isolated from a tidal flat.

A lipolytic, Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated BPTF-M16T, was isolated from tidal flat sediment on the Yellow Sea in the Republic of Korea. Strain BPTF-M16T grew optimally at 30 °C and in the presence of 2.0-3.0 % (w/v) NaCl. A phylogenetic tree of 16S rRNA gene sequences showed that strain BPTF-M16T fell within the clade comprising the type strains of Altererythrobacter species. Strain BPTF-M16T exhibited 16S rRNA gene sequence similarity values of 98.0 and 97.1 % to the type strains of Altererythrobacterishigakiensis and Altererythrobactermarinus, respectively, and of less than 97.0 % to the type strains of the other recognized species. Strain BPTF-M16T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids detected in strain BPTF-M16T were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid and one unidentified glycolipid. Mean DNA-DNA relatedness values of strain BPTF-M16T with the type strains of A. ishigakiensis and A. marinus were 22 and 13 %, respectively. The average nucleotide identity value between strain BPTF-M16T and the type strain of A. ishigakiensis was 76.80 %. Differential phenotypic properties, together with the phylogenetic and genetic data, revealed that strain BPTF-M16T is separated from recognized Altererythrobacter species. On the basis of the data presented here, strain BPTF-M16T is considered to represent a novel species of the genus Altererythrobacter, for which the name Altererythrobacter insulae sp. nov. is proposed. The type strain is BPTF-M16T (=KCTC 62421T=KACC 19609T=NBRC 113190T).

Corrigendum: An ethnically relevant consensus Korean reference genome is a step towards personal reference genomes.

This corrects the article DOI: 10.1038/ncomms13637.

An ethnically relevant consensus Korean reference genome is a step towards personal reference genomes.

Human genomes are routinely compared against a universal reference. However, this strategy could miss population-specific and personal genomic variations, which may be detected more efficiently using an ethnically relevant or personal reference. Here we report a hybrid assembly of a Korean reference genome (KOREF) for constructing personal and ethnic references by combining sequencing and mapping methods. We also build its consensus variome reference, providing information on millions of variants from 40 additional ethnically homogeneous genomes from the Korean Personal Genome Project. We find that the ethnically relevant consensus reference can be beneficial for efficient variant detection. Systematic comparison of human assemblies shows the importance of assembly quality, suggesting the necessity of new technologies to comprehensively map ethnic and personal genomic structure variations. In the era of large-scale population genome projects, the leveraging of ethnicity-specific genome assemblies as well as the human reference genome will accelerate mapping all human genome diversity.

Molecular Insights into Toluene Sensing in the TodS/TodT Signal Transduction System.

TodS is a sensor kinase that responds to various monoaromatic compounds, which either cause an agonistic or antagonistic effect on phosphorylation of its cognate response regulator TodT, and controls tod operon expression in Pseudomonas putida strains. We describe a molecular sensing mechanism of TodS that is activated in response to toluene. The crystal structures of the TodS Per-Arnt-Sim (PAS) 1 sensor domain (residues 43-164) and its complex with toluene (agonist) or 1,2,4-trimethylbenzene (antagonist) show a typical ß2α3ß3 PAS fold structure (residues 45-149), forming a hydrophobic ligand-binding site. A signal transfer region (residues 150-163) located immediately after the canonical PAS fold may be intrinsically flexible and disordered in both apo-PAS1 and antagonist-bound forms and dramatically adapt an α-helix upon toluene binding. This structural change in the signal transfer region is proposed to result in signal transmission to activate the TodS/TodT two-component signal transduction system. Site-directed mutagenesis and ß-galactosidase assays using a P. putida reporter strain system verified the essential residues involved in ligand sensing and signal transfer and suggest that the Phe(46) residue acts as a ligand-specific switch.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the ß-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses.

In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines.

Genome information of Methylobacterium oryzae, a plant-probiotic methylotroph in the phyllosphere.

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.

The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein.

The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX-TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation.

Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR.

Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N-acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for N-acetylneuraminic acid catabolism, and its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus. Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P-mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P-binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.

Charting microbial phenotypes in multiplex nanoliter batch bioreactors.

High-throughput growth phenotyping is receiving great attention for establishing the genotype-phenotype map of sequenced organisms owing to the ready availability of complete genome sequences. To date, microbial growth phenotypes have been investigated mostly by the conventional method of batch cultivation using test tubes, Erlenmeyer flasks, or the recently available microwell plates. However, the current batch cultivation methods are time- and labor-intensive and often fail to consider sophisticated environmental changes. The implementation of batch cultures at the nanoliter scale has been difficult because of the quick evaporation of the culture medium inside the reactors. Here, we report a microfluidic system that allows independent cell cultures in evaporation-free multiplex nanoliter reactors under different culture conditions to assess the behavior of cells. The design allows three experimental replicates for each of eight culture environments in a single run. We demonstrate the versatility of the device by performing growth curve experiments with Escherichia coli and microbiological assays of antibiotics against the opportunistic pathogen Pseudomonas aeruginosa. Our study highlights that the microfluidic system can effectively replace the traditional batch culture methods with nanoliter volumes of bacterial cultivations, and it may be therefore promising for high-throughput growth phenotyping as well as for single-cell analyses.

Genomic makeup of the marine flavobacterium Nonlabens (Donghaeana) dokdonensis and identification of a novel class of rhodopsins.

Rhodopsin-containing marine microbes such as those in the class Flavobacteriia play a pivotal role in the biogeochemical cycle of the euphotic zone (Fuhrman JA, Schwalbach MS, Stingl U. 2008. Proteorhodopsins: an array of physiological roles? Nat Rev Microbiol. 6:488-494). Deciphering the genome information of flavobacteria and accessing the diversity and ecological impact of microbial rhodopsins are important in understanding and preserving the global ecosystems. The genome sequence of the orange-pigmented marine flavobacterium Nonlabens dokdonensis (basonym: Donghaeana dokdonensis) DSW-6 was determined. As a marine photoheterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The sequence analysis also uncovered a gene encoding an unexpected type of microbial rhodopsin containing a unique motif in addition to a proteorhodopsin gene and a number of photolyase or cryptochrome genes. Homologs of the novel rhodopsin gene were found in other flavobacteria, alphaproteobacteria, a species of Cytophagia, a deinococcus, and even a eukaryote diatom. They all contain the characteristic NQ motif and form a phylogenetically distinct group. Expression analysis of this rhodopsin gene in DSW-6 indicated that it is induced at high NaCl concentrations, as well as in the presence of light and the absence of nutrients. Genomic and metagenomic surveys demonstrate the diversity of the NQ rhodopsins in nature and the prevalent occurrence of the encoding genes among microbial communities inhabiting hypersaline niches, suggesting its involvement in sodium metabolism and the sodium-adapted lifestyle.

Gaetbulibacter lutimaris sp. nov., isolated from a tidal flat sediment.

A Gram-stain-negative, aerobic, non-flagellated, non-gliding, rod-shaped bacterial strain, D1-y4(T), was isolated from a tidal flat sediment of the South Sea in South Korea and subjected to a polyphasic study. Strain D1-y4(T) grew optimally at 25 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain D1-y4(T) belonged to the genus Gaetbulibacter, joining the type strain of Gaetbulibacter marinus, with which it exhibited 97.8 % similarity. Sequence similarities to Gaetbulibacter saemankumensis SMK-12(T) and Gaetbulibacter aestuarii KYW382(T) were 96.5 and 96.2 %, respectively. Strain D1-y4(T) contained MK-6 as the predominant menaquionone and iso-C15 : 0, iso-C15 : 1 G and anteiso-C15 : 0 as the major fatty acids. The major polar lipids were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of strain D1-y4(T) was 34.6 mol% and its mean DNA-DNA relatedness value with G. marinus KCTC 23046(T) was 7 %. The phylogenetic and genetic distinctiveness and differential phenotypic properties revealed that strain D1-y4(T) is distinguishable from the three recognized Gaetbulibacter species. On the basis of the data presented here, strain D1-y4(T) is considered to represent a novel species of the genus Gaetbulibacter, for which the name Gaetbulibacter lutimaris sp. nov. is proposed. The type strain is D1-y4(T) ( = KCTC 23716(T)  = CCUG 61504(T)).

Kangiella sediminilitoris sp. nov., isolated from a tidal flat sediment.

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped bacterial strain, BB-Mw22(T), was isolated from a tidal flat sediment of the South Sea in South Korea. It grew optimally at 30-37 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain BB-Mw22(T) belonged to the genus Kangiella and the cluster comprising Kangiella species and strain BB-Mw22(T) was clearly separated from other taxa. Strain BB-Mw22(T) exhibited 95.3-98.7 % 16S rRNA gene sequence similarity to the type strains of recognized Kangiella species. Strain BB-Mw22(T) contained Q-8 as the predominant ubiquionone and iso-C15 : 0 and iso-C11 : 0 3-OH as the major fatty acids. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and one unidentified aminolipid. The DNA G+C content of strain BB-Mw22(T) was 48.9 mol%, and its mean DNA-DNA hybridization values with Kangiella geojedonensis YCS-5(T), Kangiella japonica JCM 16211(T) and Kangiella taiwanensis JCM 17727(T) were 14-28 %. Phylogenetic and genetic distinctiveness and differential phenotypic properties revealed that strain BB-Mw22(T) is distinguishable from all recognized Kangiella species. On the basis of the data presented, strain BB-Mw22(T) is considered to represent a novel species of the genus Kangiella, for which the name Kangiella sediminilitoris sp. nov. is proposed. The type strain is BB-Mw22(T) ( = KCTC 23892(T)  = CCUG 62217(T)).

Complete genome sequence of the probiotic bacterium Bifidobacterium bifidum strain BGN4.

Bifidobacterium bifidum, a common endosymbiotic inhabitant of the human gut, is considered a prominent probiotic microorganism that may promote health. We completely decrypted the 2.2-Mb genome sequence of B. bifidum BGN4, a strain that had been isolated from the fecal sample of a healthy breast-fed infant, and annotated 1,835 coding sequences.

Construction and manipulation of a new Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone.

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

Comparative multi-omics systems analysis of Escherichia coli strains B and K-12.

BACKGROUND: Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced. RESULTS: We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions. CONCLUSIONS: This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.

Novel metagenome-derived, cold-adapted alkaline phospholipase with superior lipase activity as an intermediate between phospholipase and lipase.

A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A(1) family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg(-1), respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C(16)), tributyrin (C(4)), and 1,2-dihexanoyl-phosphatidylcholine (C(8)). Finally, MPlaG was identified as a phospholipase A(1) with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A(1) with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases.

Efficient production of polymyxin in the surrogate host Bacillus subtilis by introducing a foreign ectB gene and disrupting the abrB gene.

In our previous study, Bacillus subtilis strain BSK3S, containing a polymyxin biosynthetic gene cluster from Paenibacillus polymyxa, could produce polymyxin only in the presence of exogenously added L-2,4-diaminobutyric acid (Dab). The dependence of polymyxin production on exogenous Dab was removed by introducing an ectB gene encoding the diaminobutyrate synthase of P. polymyxa into BSK3S (resulting in strain BSK4). We found, by observing the complete inhibition of polymyxin synthesis when the spo0A gene was knocked out (strain BSK4-0A), that Spo0A is indispensable for the production of polymyxin. Interestingly, the abrB-spo0A double-knockout mutant, BSK4-0A-rB, and the single abrB mutant, BSK4-rB, showed 1.7- and 2.3-fold increases, respectively, in polymyxin production over that of BSK4. These results coincided with the transcription levels of pmxA in the strains observed by quantitative real-time PCR (qRT-PCR). The AbrB protein was shown to bind directly to the upstream region of pmxA, indicating that AbrB directly inhibits the transcription of polymyxin biosynthetic genes. The BSK4-rB strain, producing high levels of polymyxin, will be useful for the development and production of novel polymyxin derivatives.