Systematic Insertional Mutagenesis of a Streptomycete Genome: A Link Between Osmoadaptation and Antibiotic Production

Abstract

The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces up to fivefold greater antibiotic yields than the wild-type strain.[The sequence data from this study have been submitted to EMBL under accession nos. AJ565873 and AJ566337.]

Multiple allosteric effectors control the affinity of DasR for its target sites

“…Derivatives of cosmid D95A carrying Tn5062 inserted at nucleotide position þ299 of SCO4284 (transposant cosmid: D95A.2.C09) were introduced into S. coelicolor M145 by conjugation from E. coli ET12567/pUZ8002 according to the described protocol [22]. The cosmid used for gene interruption was generated using the in vitro transposition method [22] and obtained from Prof. Paul Dyson (Swansea, UK).…”

“…The cosmid used for gene interruption was generated using the in vitro transposition method [22] and obtained from Prof. Paul Dyson (Swansea, UK).…”

Novel σ B regulation modules of Gram-positive bacteria involve the use of complex hybrid histidine kinases

“…These include the type II homologues SCO5748 (OsaA) of Streptomyces coelicolor and MXAN_0712 of Myxococcus xanthus. OsaA of S. coelicolor and its putative cognate RR OsaB have been implicated to function in osmoadaptation, aerial mycelium formation and the coordination of antibiotic production (Bishop et al, 2004), while MXAN_0712 of M. xanthus was shown to be essential in fruiting body formation and sporulation (Shi et al, 2008). To obtain additional information about the potential biological role of the other RsbK homologues, especially with respect to the possible regulation of alternative sigma factors, we analysed the genomic regions surrounding RsbK-encoding genes.…”

“…Novel activation routes for s B in Gram-positive bacteria As described above, the RsbK-type HK OsaA and its putative cognate RR OsaB of Streptomyces coelicolor have been implicated in osmoadaptation, cellular differentiation and the production of antibiotics (Bishop et al, 2004). In S. coelicolor, these processes are also controlled by a seemingly complex network of s B -like sigma factors (Cho et al, 2001;Lee et al, 2005;Viollier et al, 2003).…”

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces

“…Mutant strains were confirmed by Southern blotting, using chromosomal DNA digested with SalI. Southern hybridization was carried out by established procedures using the digoxigenin-labelled 3442-bp Tn5062 PvuII fragment from plasmid pQM5062 (Bishop et al 2004) as a probe.…”