Characterisation of conformational and functional features of alkyl hydroperoxide reductase E-like protein.

Alkyl hydroperoxide reductase E (AhpE) is a member of the peroxidase family of enzymes that catalyse the reduction of peroxides, however its structural and functional roles are still unclear in details. In this study, we used the Thermococcus kodakarensis AhpE-like protein as a model to investigate structure-function relationships including the molecular properties of DNA binding activity. Multiple sequence alignment, structural comparison and biochemical analyses revealed that TkAhpE includes conserved peroxidase residues in the active site, and exhibits peroxidase activity with structure-dependent holdase chaperone function. Following electrophoretic mobility shift assays and electron microscopy analysis demonstrated distinctive binding features of TkAhpE to the DNA showing that their dimeric conformer can bind to the double-stranded DNA, but not to the single-stranded DNA, indicating its striking molecular features to double-stranded DNA-specific interactions. Based on our results, we provided that TkAhpE is a multifunctional peroxidase displaying structure-dependent molecular chaperone and DNA binding activities.

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.

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.

Lysobacter capsici sp. nov., with antimicrobial activity, isolated from the rhizosphere of pepper, and emended description of the genus Lysobacter.

The taxonomic position of a novel bacterial strain, YC5194(T), with antimicrobial activity, isolated from the rhizosphere of pepper in Jinju, South Korea, was studied using a polyphasic approach. Cells of the strain were Gram-negative, rod-shaped, facultative anaerobes. It grew at a temperature of 15-37 degrees C (optimum 28 degrees C). Growth of the strain occurred between pH 5.5 and 8.5, with an optimum of pH 7.0-7.5. The strain inhibited mycelial growth of Pythium ultimum, Colletotrichum gloeosporioides, Fusarium oxysporum, Botrytis cinerea, Rhizoctonia solani and Botryosphaeria dothidea and growth of Bacillus subtilis. The G+C content of the total DNA was 65.4 mol%. The 16S rRNA gene sequence of the strain was most closely related to species of the genus Lysobacter (<94.0 to >99.0 % sequence similarity). Chemotaxonomic data (major quinone, Q-8; major polar lipids, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyl-N-methylethanolamine; major fatty acids, iso-C(15 : 0), summed feature 3, C(16 : 0), iso-C(17 : 1)omega9c and C(18 : 1)omega7c) supported the affiliation of strain YC5194(T) to the genus Lysobacter. Phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA hybridization data and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain YC5194(T) from species of Lysobacter with validly published names. Strain YC5194(T) therefore represents a novel species, for which the name Lysobacter capsici sp. nov. is proposed. The type strain is YC5194(T) (=KCTC 22007(T) =DSM 19286(T)).

Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681.

Fusaricidin, a peptide antibiotic consisting of six amino acids, has been identified as a potential antifungal agent from Paenibacillus polymyxa. Here, we report the complete sequence of the fusaricidin synthetase gene (fusA) identified from the genome sequence of a rhizobacterium, P. polymyxa E681. The gene encodes a polypeptide consisting of six modules in a single open-reading frame. Interestingly, module six of FusA does not contain an epimerization domain, which suggests that the sixth amino acids of the fusaricidin analogs produced by P. polymyxa E681 may exist as an l-form, although all reported fusaricidins contain d-form alanines in their sixth amino acid residues. Alternatively, the sixth adenylation domain of the FusA may directly recognize the d-form alanine. The inactivation of fusA led to the complete loss of antifungal activity against Fusarium oxysporum. LC/MS analysis confirmed the incapability of fusaricidin production in the fusA mutant strain, thus demonstrating that fusA is involved in fusaricidin biosynthesis. Our findings suggested that FusA can produce more than one kind of fusaricidin, as various forms of fusaricidins were identified from P. polymyxa E681.