Effect of protein phosphatase gene sppA deletion on hyphal growth in Streptomyces coelicolor when combined with different protein kinase gene afsK mutations

Abstract: Streptomyces species are known for their bacteria-untypical growth and sporulation. Growth by tip extension building up a mycelium consisting of long branching hyphae is a highly polar and regulated process. An essential protein invalved in hyphal growth and branching regulation is DiviVA. In S. coelicolor branching and growth is regulated by phosphorylation of DiviVA by the serine/tlu•eonine lcinase AfsK. The phosphatase SppA dephosphorylates DiviVA and leads to less branching growth, an interaction to be further investigated in this project. The aim of this project was to combine a sppA knockout mutation with different afsK modifications, such as deletion and overexpression mutants. A cosmid carrying a new ilsppA::vph allele was constructed using the Å-Red system in E. coli. The allele was transfened to S. coelicolor and recombinant strains with replacement of the sppA gene were selected. Of special interest was the ilsppA ilafsK double lu1ockout to reveal possible additional DiviVA phosphorylation due to other kinases and to check if the wild type colony size phenotype (previously small colonies were observed in ilsppA strains) would be regained. The obtained strains were examined for growth phenotypes and phosphorylation patterns of DiviVA were visualized by Western blotting. sppA knockout mutants were tested for increased sensitivity to overexpression of afsK. The obtained results suppmi the previously raised hypothesis, that SppA and AfsK are direct counteractors on DiviVA phosphorylation. The sppA afsK double knockout mutant pmily regains the wild type colony size. Sensitivity to overexpression of ajsK seems to be strongly increased by an sppA knockout. As time was limited the results are preliminary and further experiments should be carried out with the generated strains to confirm and extend the results.