The MreB-Like Protein Mbl ofStreptomyces coelicolorA3(2) Depends on MreB for Proper Localization and Contributes to Spore Wall Synthesis

Abstract

Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ⌬mreB ⌬mbl double mutant was viable. ⌬sco6166 had a wild-type phenotype. ⌬mreB, ⌬mbl, and ⌬mreB ⌬mbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the ⌬mbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.The peptidoglycan (PG) layer, consisting of long glycan strands cross-linked by short peptides, is a major determinant of bacterial cell shape (51). Most species with a complex, nonspherical morphology contain the actin-like MreB protein, which belongs to the HSP70-actin-sugar kinase (ASHKA) superfamily of proteins (4, 48). MreB was shown to polymerize into a dynamic helical filament underneath the cytoplasmic membrane spanning the long axis of the cells (11,17,25,30,33). In rod-shaped bacteria, MreB is thought to interact with other proteins to position a cell wall-synthesizing complex at the lateral cell wall (16,17,32). The incorporation of new PG at the lateral wall results in cell elongation, thus determining rod-shaped morphology. Gram-negative bacteria seem to have a single mreB gene, usually in an operon with mreC and mreD.

Genetic Analysis of SCO2997 , Encoding a TagF Homologue, Indicates a Role for Wall Teichoic Acids in Sporulation of Streptomyces coelicolor A3(2)

“…The spores of EMK2997 and EMK2584 were significantly shorter than those of M145. While S. coelicolor M145 produced uniform ovoid spores of 1.27 Ϯ 0.16 m by 0.68 Ϯ 0.07 m in size, mutant spores were shorter and showed a wider variation in length, from 0.6 m up to 2.4 m. The mutant phenotype was even more severe when the mutants were grown on SM agar supplemented with 10.3% sucrose, as has been also reported for ftsI and ftsW mutants (3) or mutants affected in genes of the SSSC (12,19). Genetic complementation of the mutants by integrating an intact copy of tagF into the C31 attachment site of EMK2997 or by introducing plasmid pGM2584 (Table 1) carrying SCO2584-His 6 under the control of the thiostreptoninducible P tipA promoter into EMK2584 partially restored spore dimensions ( Fig.…”

“…S1A in the supplemental material). Frequently, the mutant spores germinated prematurely in the spore chain, a phenotype characteristic for mre mutants of S. coelicolor (12,19,24). After 4 days of incubation on SM agar at 30°C, spore sizes (n ϭ 750) were measured with the AnalySIS program and compared by box plot analysis (Fig.…”

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…Besides the tubulin homolog FtsZ and the actin-like proteins MreB and Mbl (320,321), a large number of proteins with coiled-coil structural elements occur in these bacteria, and evidence is accumulating regarding their important role in growth, cell shape, and morphogenesis (319,(322)(323)(324). The protein FilP forms intermediate filament-like structures that contribute to mechanical stress resistance (322).…”

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

“…In sharp contrast, streptomycetes grow by tip extension and hyphal branching. 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).…”