Investigations of siderophore and tetronic acid biosynthesis in streptomyces scabies 87.22

Abstract

Streptomyces are Gram-positive bacteria, usually found living within the soil and they are saphrophytes. Among this class of bacteria are some plant pathogenic species, which cause infection of the roots or the tubers of some plants. The model Streptomycete plant pathogen is Streptomyces scabies; this infects root crops, such as potato or radish and is a known cause of scab disease. Most Streptomyces species are producers of secondary metabolites, many of which possess important biological activities, such as antibacterial, iron-chelating, anticancer or immunosuppressant. One group of these secondary metabolites are called siderophores. These are small organic molecules, which can chelate ferric iron. The iron in the environment is mainly present as iron (III) hydroxide, which is not very water soluble and cannot, therefore, be taken up directly by microorganisms. Some bacteria solve this problem through production of siderophores. The siderophores are released into the environment by the microorganisms to chelate iron (III) from the environment and transport it into the cell across the cell membrane. Iron is required for many life processes. Analysis of the Streptomyces scabies genome sequence resulted in the identification of gene clusters predicted to direct the biosynthesis of known siderophores, e.g. desferrioxamines and pyochelin, as well as, potentially novel siderophores. A gene inactivation and comparative metabolic profiling approach has been employed to identify the metabolic products of these gene clusters. A PCR-targeting method was used to replace part of or whole genes in the S. scabies 87.22 putative secondary metabolite gene clusters. An internal fragment of the scabichelin biosynthetic gene scab85471 and the putative S. scabies desC gene were deleted using this method. The scabichelin and desC gene mutants were subsequently analysed by LC-MS allowing confirmation of the function of the genes investigated. Production of scabichelin by S. scabies 87.22 wild type was analysed by comparing it with the authentic standard. The chemical and genetic complementation of the Δ desC mutant was carried out to establish the involvement of the desC gene in the biosynthesis of desferrioxamines. The S. scabies 87.22 cryptic tetronate biosynthetic gene cluster predicted to encode a novel agglomerin-like product, which could potentially be involved in plant pathogenicity was also investigated. The expression of the gene cluster was first analysed using reverse transcriptase PCR (RT-PCR) which was carried out on the total RNA isolated from the wild type S. scabies. Following this, an attempt was made to disrupt the scab63021 gene, a putative transcriptional activator of the cryptic tetronate-like cluster in the S. scabies genome. Transcriptional analysis of the wild type S. scabies and the putative Δscab63021 mutant genomes did not show any difference in the expression of the tetronate genes between the wild type strain and the Δ scab63021 mutant