Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067

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.     

Molecular Cloning,Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.     

Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.     

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.     

Construction and use of a new vector/transposon, pLBT::mim-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium

Abstract The previously described pLOFKm transposon delivery plasmid (J. Bacteriol. (1990) 172, 6557–6567) was engineered such that a promoterless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.