Soo-Keun Choi

Identification of a Polymyxin Synthetase Gene Cluster of Paenibacillus polymyxa and Heterologous Expression of the Gene in Bacillus subtilis

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated pmxA, pmxB, pmxC, pmxD, and pmxE. The pmxC and pmxD genes are similar to genes that encode transport proteins, while pmxA, pmxB, and pmxE encode polymyxin synthetases. The insertional disruption of pmxE led to a loss of the ability to produce polymyxin. Introduction of the pmx gene cluster into the amyE locus of the Bacillus subtilis chromosome resulted in the production of polymyxin in the presence of extracellularly added L-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the pmx gene cluster is responsible for polymyxin biosynthesis.

Genome Sequence of the Polymyxin-Producing Plant-Probiotic Rhizobacterium Paenibacillus polymyxa E681

Paenibacillus polymyxa E681, a spore-forming, low-G+C, Gram-positive bacterium isolated from the rhizosphere of winter barley grown in South Korea, has great potential for agricultural applications due to its ability to promote plant growth and suppress plant diseases. Here we present the complete genome sequence of P. polymyxa E681. Its 5.4-Mb genome encodes functions specialized to the plant-associated lifestyle and characteristics that are beneficial to plants, such as the production of a plant growth hormone, antibiotics, and hydrolytic enzymes.

Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis.

Bacillus thuringiensis produces crystal proteins (Cry) that account for up to 25% of the dry cell weight during the stationary phase. The high-level expression and stationary phase-specific autoinduction of the cry gene led to development of a cry promoter-based Bacillus expression system. Among the various cry promoters, cry3Aa promoter was selected by comparing the lacZ expression levels in Bacillus subtilis. An extracellular enzyme cellulase was highly upregulated during the stationary phase while under control of the cry3Aa promoter. Improvement of the cry3Aa promoter was obtained by modification of the promoter sequence. Specifically, a 5-fold increase in lacZ expression was obtained by changing both the -35 and -10 boxes of the cry3Aa promoter to the consensus sequence of the sigma(A)-dependent promoter of B. subtilis. The modified cry3Aa promoter produced a significantly higher yield of AprE, which suggests that the promoter may be useful for high-level protein expression in B. subtilis.

Citrinin, a mycotoxin from Penicillium citrinum, plays a role in inducing motility of Paenibacillus polymyxa

Paenibacillus polymyxa, a Gram-positive low-G+C spore-forming soil bacterium, belongs to the plant growth-promoting rhizobacteria. The swarming motility of P. polymyxa strain E681 was greatly induced by a secondary metabolite, citrinin, produced by Penicillium citrinum KCTC6549 in a dose-dependent manner at concentrations of 2.5-15.0 microg mL(-1) on tryptic soy agar plates containing 1.0% (w/v) agar. Flagellum staining showed that citrinin activated the production of flagella by P. polymyxa. This result was supported by reverse transcriptase-PCR analysis of gene expression, which showed increased transcriptional levels of sigD and hag homologues of P. polymyxa E681 in the presence of citrinin. The results presented here show that a mycotoxin, citrinin, has a newly identified function of inducing bacterial motility by transcriptional activation of related genes. This finding contributes to our understanding of the interactions between bacteria and fungal strains in nature.

Analysis of tnrA Alleles Which Result in a Glucose-Resistant Sporulation Phenotype in Bacillus subtilis

Bacillus subtilis cells cannot sporulate in the presence of catabolites such as glucose. During the analysis of Tn10-generated mutants, we found that deletion of the C-terminal region of the tnrA gene, which encodes a global regulator that positively regulates a number of genes in response to nitrogen limitation, results in a catabolite-resistant sporulation phenotype. Analyses of nrg-lacZ and nasB-lacZ, which are activated by TnrA under nitrogen limitation, showed that C-terminally truncated TnrA activates nitrogen-regulated genes constitutively. The relief of catabolite repression of sporulation may result from the uncontrolled expression of the TnrA-regulated genes.

Regulation of pho Regulon Gene Expression by the Carbon Control Protein A, CcpA, in Bacillus subtilis

Bacterial regulons involved in carbon, nitrogen and phosphorus metabolism must interact for purposes of coordination, but the mechanisms involved are not understood. We here report that the carbon control pro-tein-A (CcpA) of Bacillus subtilis, primarily concerned with carbon metabolism, influences expression of various phosphorus (pho) regulon genes including the two alkaline phosphatase structural genes, phoA and phoB. The directions and magnitudes of the effects of glucose and the loss of CcpA on these two genes depend on growth conditions, but they always correlate inversely. Absolute expression levels of phoA and phoB depend on a rich nitrogen source, and gene activation by a fermentable substrate such as glucose depends on the presence of a respiratory substrate such as succinate. We show that these CcpA-dependent glucose effects can be explained by the effects of glucose and CcpA acting on the phoPR operon. Although a good CcpA-binding site (CRE) is found in the control region of the phoPR operon, direct regulation of phoPR gene expression by CcpA via this CRE could not account for the effects of glucose and CcpA on phoA and phoB gene expression. We conclude that CcpA exerts indirect control over the pho regulon by a mechanism that involves CcpA and PhoRP but does not involve the phoPR operon CRE.

Mechanism of CcpA-mediated glucose repression of the resABCDE operon of Bacillus subtilis.

The resABCDE operon of Bacillus subtilis encodes a three-protein complex involved in cytochrome c biogenesis as well as the ResE sensor kinase and the ResD response regulator that control electron transfer and other functions in response to oxygen availability. We have investigated the mechanism of CcpA-mediated control of res operon expression which occurs maximally in the stationary phase of growth. Two CcpA-binding (CRE) sites were found in the res operon, one (CRE1) in the control region in front of the resA promoter, the other (CRE2) in the resB structural gene. Both CRE sites proved to be essential for full CcpA-mediated glucose repression of res operon expression. We propose that both looping and road block mechanisms are involved in res operon control by CcpA.

Genome Sequence of Lysinibacillus sphaericus Strain KCTC 3346T

ABSTRACT Lysinibacillus sphaericus is a heterogeneous species that includes strains that produce mosquitocidal toxin proteins. Herein, we report the 4.56-Mb draft genome sequence of the nonpathogenic L. sphaericus strain KCTC 3346T, which provides clues for the phylogenetic reassessment of L. sphaericus species and an understanding of its physiological properties.

Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

Genome Sequence of the Acrystalliferous Bacillus thuringiensis Serovar Israelensis Strain 4Q7, Widely Used as a Recombination Host

ABSTRACT Bacillus thuringiensis serovar israelensis is well known for its mosquitocidal activity and has long been used as a biopesticide. Herein, we present the genome sequence of B. thuringiensis serovar israelensis strain 4Q7, a plasmid-cured derivative with higher transformation efficiency than wild types.