In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.hyphal growth | protein phosphorylation | peptidoglycan | cytoskeleton | tip extension

Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.Gram-positive bacteria of the genus Streptomyces grow by tip extension and form branched hyphae and mycelia (8,11,12). This polarized cell wall growth is strikingly different from the mode of growth of, e.g., Escherichia coli and Bacillus subtilis, which like most rod-shaped bacteria extend the cell and acquire their rod shape by intercalatory insertion of new peptidoglycan units along the lateral wall (5, 6). This is dependent on the actin-like MreB proteins, which form helical filaments extending along the cell and acting via interaction with membrane proteins to organize the cell wall assembly (1,2,16,19). In contrast, Streptomyces tip extension appears to occur by an mreB-independent mechanism (22) and is also independent of FtsZ and cell division (23). The Streptomyces coelicolor genome contains two mreB genes, but they are involved primarily in sporulation and have no overt impact on tip extension in the vegetative mycelium (22; G. Muth, University of Tübingen, Germany, personal communication). In fact, most rod-shaped relatives of Streptomyces within the phylum Actinobacteria, like mycobacteria and corynebacteria, lack mreB genes and assemble their cell walls at the cell poles (3,5,15,24).This mreB-independent and polarized growth in Actinobacteria involves the coiled-coil protein DivIVA. In S. coelicolor, DivIVA is essential for growth and accumulates at growing hyphal tips, and the effects of partial depletion and ectopic overexpression revealed a strong impact on tip extension and cell shape determination (10). Among other Actinobacteria, the DivIVA orthologues, also named antigen 84 and Wag31, in Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum are polarly localized and appear to be essential and, when overproduced, have a very similar effect on cell shape to that seen in S. coelicolor (17,24,25). Recently, DivIVA was found to be required for polar cell elongation and acquisition of rod shape in C. glutamicum and M. smegmatis (17,20). Furthermore, Streptomyces and Mycobacterium DivIVA could restore polar growth to a C. glutamicum strain depleted for DivIVA, while orthologues from the phylum Firmicutes (e.g., Bacillus subtilis) could not (20) and are known to be associated with different cellular functions (9,21,26,29). While these findings suggest a role for Streptomyces DivIVA in tip extension, its exact function is not known. In this study, we have investigated the subcellular targeting of S. coelicolor DivIVA and its involvement in the establishment of tip extension during hyphal branching.DivIVA is a molecular marker of new branch sites in S. coelicolor. Apart from the striking apical localization of S. coelicolor DivIVA, o...

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