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The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin.

ABSTRACT: Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study, the ocfC gene, which is located downstream of ocfD and upstream of the ocfB gene in the ocf gene cluster, was examined. Antifungal activity of the ocfC gene mutant MS14KC1 was reduced against the indicator fungus Geotrichum candidum compared with that of the wild-type strain. Furthermore, the analysis of the protein sequence suggests that the ocfC gene encodes a glycosyltransferase. Biochemical analyses using nuclear magnetic resonance (NMR) and mass spectroscopy revealed that the ocfC mutant produced the occidiofungin without the xylose. The purified ocfC mutant MS14KC1 product had a level of bioactivity similar to that of the wild-type product. The revertant MS14KC1-R of the ocfC mutant produced the same antifungal activity level on plate assays and the same antifungal compound based on high-performance liquid chromatography (HPLC) and mass spectroscopy analysis as wild-type strain MS14. Collectively, the study demonstrates that the ocfC gene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity against Candida species. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi, and the finding could also simplify the chemical synthesis of occidiofungin variants.

SUBMITTER: Chen KC

PROVIDER: S-EPMC3623131 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin.

Chen Kuan-Chih KC Ravichandran Akshaya A Guerrero Adam A Deng Peng P Baird Sonya M SM Smith Leif L Lu Shi-En SE

Applied and environmental microbiology 20130222 9

Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study, the ocfC gene, which is located downstream of ocfD and upstream of the ocfB gene in the ocf gene cluster, w ...[more]

PMID: 23435879

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Project description:Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.

Project description:Burkholderia contaminans MS14 shows significant antimicrobial activities against plant and animal pathogenic fungi and bacteria. The antifungal agent occidiofungin produced by MS14 has great potential for development of biopesticides and pharmaceutical drugs. However, the use of Burkholderia species as biocontrol agent in agriculture is restricted due to the difficulties in distinguishing between plant growth-promoting bacteria and the pathogenic bacteria. The complete MS14 genome was sequenced and analyzed to find what beneficial and virulence-related genes it harbors. The phylogenetic relatedness of B. contaminans MS14 and other 17 Burkholderia species was also analyzed. To research MS14's potential virulence, the gene regions related to the antibiotic production, antibiotic resistance, and virulence were compared between MS14 and other Burkholderia genomes. The genome of B. contaminans MS14 was sequenced and annotated. The genomic analyses reveal the presence of multiple gene sets for antimicrobial biosynthesis, which contribute to its antimicrobial activities. BLAST results indicate that the MS14 genome harbors a large number of unique regions. MS14 is closely related to another plant growth-promoting Burkholderia strain B. lata 383 according to the average nucleotide identity data. Moreover, according to the phylogenetic analysis, plant growth-promoting species isolated from soils and mammalian pathogenic species are clustered together, respectively. MS14 has multiple antimicrobial activity-related genes identified from the genome, but it lacks key virulence-related gene loci found in the pathogenic strains. Additionally, plant growth-promoting Burkholderia species have one or more antimicrobial biosynthesis genes in their genomes as compared with nonplant growth-promoting soil-isolated Burkholderia species. On the other hand, pathogenic species harbor multiple virulence-associated gene loci that are not present in nonpathogenic Burkholderia species. The MS14 genome as well as Burkholderia species genome show considerable diversity. Multiple antimicrobial agent biosynthesis genes were identified in the genome of plant growth-promoting species of Burkholderia. In addition, by comparing to nonpathogenic Burkholderia species, pathogenic Burkholderia species have more characterized homologs of the gene loci known to contribute to pathogenicity and virulence to plant and animals.

Project description:The Burkholderia cepacia complex is a group of opportunistic pathogens that cause both severe acute and chronic respiratory infections. Due to their large genomes containing multiple intrinsic and acquired antimicrobial resistance mechanisms, treatment is often difficult and prolonged. One alternative to traditional antibiotics for treatment of bacterial infections is bacteriophages. Therefore, the characterization of bacteriophages infective for the Burkholderia cepacia complex is critical to determine their suitability for any future use. Here, we describe the isolation and characterization of novel phage, CSP3, infective against a clinical isolate of Burkholderia contaminans. CSP3 is a new member of the Lessievirus genus that targets various Burkholderia cepacia complex organisms. Single nucleotide polymorphism (SNP) analysis of CSP3-resistant B. contaminans showed that mutations to the O-antigen ligase gene, waaL, consequently inhibited CSP3 infection. This mutant phenotype is predicted to result in the loss of cell surface O-antigen, contrary to a related phage that requires the inner core of the lipopolysaccharide for infection. Additionally, liquid infection assays showed that CSP3 provides suppression of B. contaminans growth for up to 14 h. Despite the inclusion of genes that are typical of the phage lysogenic life cycle, we saw no evidence of CSP3's ability to lysogenize. Continuation of phage isolation and characterization is crucial in developing large and diverse phage banks for global usage in cases of antibiotic-resistant bacterial infections. IMPORTANCE Amid the global antibiotic resistance crisis, novel antimicrobials are needed to treat problematic bacterial infections, including those from the Burkholderia cepacia complex. One such alternative is the use of bacteriophages; however, a lot is still unknown about their biology. Bacteriophage characterization studies are of high importance for building phage banks, as future work in developing treatments such as phage cocktails should require well-characterized phages. Here, we report the isolation and characterization of a novel Burkholderia contaminans phage that requires the O-antigen for infection, a distinct phenotype seen among other related phages. Our findings presented in this article expand on the ever-evolving phage biology field, uncovering unique phage-host relationships and mechanisms of infection.

Dataset Information

The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin.

Publications

The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin.

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