Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth inStreptomyces coelicolor

Abstract

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...

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

“…In summary, the function of AfsK in controlling polar growth and branching is not obviously related to the previously inferred role of AfsK in secondary metabolism. Overall, our findings show that communication between the polarity determinant DivIVA and the cell wall biosynthetic machinery is bidirectional, with DivIVA directing cell wall synthesis (11), and the biosynthetic machinery communicating back to DivIVA via AfsK-mediated phosphorylation. All three components-the cell wall biosynthetic machinery, AfsK, and DivIVAlocalize to growing hyphal tips.…”

“…Deconvolution of fluorescence images used the iterative restoration algorithm in Volocity 3DM (Perkin-Elmer) and a calculated point spread function, and was carried out on z-stacks of more than 50 images with 0.2-μm spacing, captured with a 100× NA 1.4 lens. Live-cell time-lapse microscopy was performed as described previously (11).…”

“…This apical mode of growth is independent of MreB (9), and instead depends on the coiled-coil protein DivIVA, which is localized in large assemblies at growing hyphal tips (10). divIVA is essential for growth, and overexpression of divIVA is sufficient to trigger hyper-branching, showing that DivIVA is a key determinant of polarized growth in Streptomyces (10,11). Together with the nonessential coiled-coil protein Scy (12), DivIVA forms an apical multiprotein complex, here termed the bacterial polarisome by analogy with the polarisome complex that directs cell polarity in yeasts and filamentous fungi (13).…”

“…It was previously demonstrated that small foci of DivIVA mark the sites of new branches before visible outgrowth occurs (11), although it remained unclear how such polarity marks are established (5). However, we have recently shown that the foci of DivIVA that trigger branching are primarily created by a unique polarisome splitting mechanism, in which the apical polarisome, visualized as a tip focus of DivIVA-EGFP, splits to deposit a small daughter polarisome, which is left behind on the lateral membrane as the tip grows away (14).…”

Coiled-coil protein Scy is a key component of a multiprotein assembly controlling polarized growth inStreptomyces

“…Polarized hyphal extension in S. coelicolor depends on the coiled-coil protein DivIVA (8,13). The divIVA gene is essential in S. coelicolor, where either partial depletion or overproduction of DivIVA resulted in aberrant branching and altered hyphal characteristics (8,14). DivIVA-Egfp localized to growing hyphal tips and also to sites in the lateral wall, marking future branch points before the appearance of the new lateral tip (14).…”