We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.
Gene expression Biosynthesis HPLC/MS analysis E. coli inhibition
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We thank David. L. Coplin for valuable comments on the manuscript.
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