PCR screening reveals considerable unexploited biosynthetic potential of ansamycins and a mysterious family of AHBA-containing natural products in actinomycetes.

AIMS: Ansamycins are a family of macrolactams that are synthesized by type I polyketide synthase (PKS) using 3-amino-5-hydroxybenzoic acid (AHBA) as the starter unit. Most members of the family have strong antimicrobial, antifungal, anticancer and/or antiviral activities. We aimed to discover new ansamycins and/or other AHBA-containing natural products from actinobacteria. METHODS AND RESULTS: Through PCR screening of AHBA synthase gene, we identified 26 AHBA synthase gene-positive strains from 206 plant-associated actinomycetes (five positives) and 688 marine-derived actinomycetes (21 positives), representing a positive ratio of 2·4-3·1%. Twenty-five ansamycins, including eight new compounds, were isolated from six AHBA synthase gene-positive strains through TLC-guided fractionations followed by repeated column chromatography. To gain information about those potential ansamycin gene clusters whose products were unknown, seven strains with phylogenetically divergent AHBA synthase genes were subjected to fosmid library construction. Of the seven gene clusters we obtained, three show characteristics for typical ansamycin gene clusters, and other four, from Micromonospora spp., appear to lack the amide synthase gene, which is unusual for ansamycin biosynthesis. The gene composition of these four gene clusters suggests that they are involved in the biosynthesis of a new family of hybrid PK-NRP compounds containing AHBA substructure. CONCLUSIONS: PCR screening of AHBA synthase is an efficient approach to discover novel ansamycins and other AHBA-containing natural products. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that the AHBA-based screening method is a useful approach for discovering novel ansamycins and other AHBA-containing natural products from new microbial resources.

Polymorphism of metabolic genes and susceptibility to occupational chronic manganism.

In this study we investigated genetic polymorphisms of five metabolizing genes and their association with occupational chronic manganism. We recruited 49 patients with chronic manganism and 50 unrelated healthy control subjects who were welders and ferromanganese smelters and occupationally exposed to manganese dust and fume in the same workshops from three metallurgical industries. The controls were matched to the cases by sex, age, cigarette and alcohol intake, as well as the manganese exposure duration. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the cytochrome P450 2D6L gene (CYP2D6L) and the NAD(P)H:quinone oxidoreductase gene (NQO1). Allele-specific PCR was used to detect the cytochrome P450 1A1 gene (CYP1A1), and the glutathione-S-transferase mu and theta genes (GSTM and GSTT). The frequency of polymorphic alleles, a mutation of CYP2D6L, was significantly lower in patients with chronic manganism (16.3%) than in controls (29.0%). Individuals with the homozygote polymorphism (L/L) of CYP2D6 had a 90% decreased risk of chronic manganism compared with the wild-type (Wt/Wt) (odds ratio =0.10, 95% confidence interval = 0.01-0.82). A significant association between the CYP2D6 genotype subgroup and the latency of chronic manganese poisoning was also found. Patients who had homozygous (L/L) or heterozygous (Wt/L) mutant alleles developed manganism an average of 10 years later than those who were homozygous wildtype (Wt/Wt). However, the allele and genotype frequencies of CYP1A1 and NQO1 genes were distributed similarly in cases and controls. In addition, no difference in the frequencies of GSTM1 and GSTT1 null genotypes were observed between cases and controls. The results suggest that CYP2D6L gene polymorphism might influence susceptibility to manganese-induced neurotoxicity. However, because of limited sample size, our results should be validated in large-scale studies.

Single-strand conformation polymorphism analysis of RT-PCR products of UK isolates of barley yellow mosaic virus.

Single-strand conformation polymorphism (SSCP) analysis of the bipartite genomes of several UK isolates of barley yellow mosaic virus (Ba YMV) was done using fragments of cDNA amplified by RT-PCR. Isolates differed in their SSCP patterns in several regions, but in no case was the pattern able to distinguish between common and resistance-breaking strains. In regions where the nucleotide sequences of UK isolates had been determined, there was no simple relationship between numbers of nucleotide differences and SSCP patterns: differences of only 2 or 3 nucleotides (nt) gave different SSCP patterns, whereas differences of as many as 29 nt did not. Although SSCP analysis has some potential as a rapid and sensitive tool for distinguishing virus isolates, differences detected do not necessarily relate to biological properties and the results are highly dependent on gel conditions.

Molecular characterisation of UK isolates of barley yellow mosaic bymovirus.

Several isolates of barley yellow mosaic virus (BaYMV) from different sites in the UK, including some that were virulent on European resistant winter barley cultivars (resistance-breaking strain: BaYMV-2) and some that were not, were examined by RT-PCR, restriction mapping and sequencing of selected parts of the virus genome. Nucleotide and predicted amino acid sequences were determined for the 5'-terminal region, part of the NIa coding region and the coat protein coding region on RNA 1 and an area at the N-terminus of the 70-kDa protein coding region on RNA 2. The sequences differed from those previously reported for a BaYMV isolate from Japan and for two German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences and the few, non-consecutive, nucleotide differences detected were probably not significant and were insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping of RNA 2 cDNA again showed no consistent strain-related differences. The differences previously reported between the two German isolates are probably not strain-related.