Forkhead-associated proteins genetically linked to the serine/threonine kinase PknB regulate carbon flux towards antibiotic biosynthesis in Streptomyces coelicolor.
Bottom Line: Here we report functional analysis of pknB and two linked genes, fhaAB, encoding forkhead-associated (FHA) domain proteins that are part of a highly conserved gene locus in actinobacteria.FhaAB are candidate interacting partners of PknB and loss of their function resulted in deregulation of central carbon metabolism, with carbon flux diverted to synthesis of the antibiotic actinorhodin.The results indicate that inactivation of FHA 'brake' proteins can potentially amplify the function of STPKs and, in this case, provide a means to overproduce antibiotics.
Affiliation: Institute of Life Science, Swansea University, Singleton Park, Swansea SA28PP, UK.Show MeSH
Related in: MedlinePlus
Mentions: The conserved actinobacterial pknB gene cluster contains two neighbouring genes, SCO3843 and 3844, encoding forkhead‐associated (FHA) proteins, hereafter termed fhaA and fhaB. It is emerging that FHA proteins are typical targets for phosphorylation by actinobacterial Ser/Thr kinases with, for example, mycobacterial PknB phosphorylating an orthologue of FhaA (Grundner et al., 2005). FHA proteins are believed to function as mediators of Ser/Thr kinase signalling. Thus, we investigated the consequences of loss of function of both fhaA and fhaB by constructing a double mutant, replacing the majority of the coding sequences for both genes by a hygromycin resistance gene. As with the pknB mutant, the fhaAB double mutant resembled the parental strain when grown on MS medium but exhibited precocious sporulation and antibiotic production on NE medium (Fig. 5A). Genetic complementation of the mutant with plasmid pSEF2, containing the fhaAB genes, restored the parental phenotype to the mutant (Fig. 5A). The yield of actinorhodin was quantified during growth in submerged culture, revealing that the fhaAB mutant produced approximately double the yield of the pknB mutant and eight times the yield of the parental strain (Fig. 2), but that growth rate was unaffected. The mutants also differed in that addition of osmolyte had no significant effect on the yields of the fhaAB mutant. As the function of the pknB gene cluster in other actinobacteria is primarily associated with growth and morphology, the observed accelerated differentiation of the mutants was further explored by examining cell morphologies. No gross differences could be detected between the substrate and aerial hyphae or spore chains of the parental strain M145 and the pknB mutant. In contrast, a large proportion of the substrate hyphae of the fhaAB mutant assembled as cords of between two and four hyphae growing together (Fig. 5B). There was no evidence of hyphal fusion as the hyphae growing in cords retained their individual cell walls, stained by fluoroscein‐conjugated wheat germ agglutinin. These cords were not observed in the complemented fhaAB strain containing plasmid pSEF2. Staining with fluorescence‐labelled vancomycin revealed no differences in the frequency of hyphal cross‐walls between the mutant and wild‐type (results not shown). We then examined the substrate hyphal cords of the mutant using atomic force microscopy. The topographic images obtained revealed up to 1 µm wide hyphae in the parental strain. In contrast, many hyphae of the fhaAB mutant were between 2 and 3 µm thick, consistent with cords comprised of multiple parallel hyphae longitudinally joined together (Fig. 5C). Apart from the dimensions, no differences could be detected between the surface of individual hyphae and that of the cords. The spore‐bearing aerial hyphae of the fhaAB mutant did not assemble as cords and the surfaces of these hyphae and resulting spore chains appeared similar to those of the parental strain when examined using AFM, decorated with characteristic chaplin and rodlin assemblies (results not shown).
Affiliation: Institute of Life Science, Swansea University, Singleton Park, Swansea SA28PP, UK.