No abstract

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.

An epidemiologic study of methicillin-resistant Staphylococcus aureus (MRSA) was conducted by antibiotype, coagulase gene typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. A total of 129 MRSA strains were isolated from 17 hospitals in the regions of the central, northern, northeastern and eastern Thailand during November 2003 -March 2004. Antimicrobial susceptibility testing with a panel of 10 antimicrobial agents showed 9 different antibiotypes. The antibiotypes 1 and 2 were the most common phenotypes with 44.2% and 35.6% of the isolates, respectively. Coagulase gene typing of MRSA strains generated 4 different genotypes: I, II, III, IV, the PCR products of which were 492±20, 654±20, 735±20 and 816±20 bp with the percentages of 1.5 (2/129), 2.3 (3/129), 82.2 (106/129) and 14 (18/129), respectively. Coagulase gene PCR-RFLPs exhibited 4 patterns: A, B, C and D, with AluI digested PCR product fragments at 220±20 and 220±20 bp (pattern A); 400±20 and 220±20 bp (pattern B); 420±20 and 220±20 bp (pattern C); and 510±20 and 220±20 bp (pattern D). The percentage values for each pattern were compatible with those from the coagulase gene typing method. The results indicated that antibiotypes 1, 2, coagulase gene type III and PCR-RFLP pattern C were the epidemic strains while the rest were sporadic strains.

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.