Spore cortex formation in <i>Bacillus subtilis</i> is regulated by accumulation of peptidoglycan precursors under the control of sigma K

Vasudevan, Pradeep; Weaver, Amy K.; Reichert, Erin D.; Linnstaedt, Sarah D.; Popham, David L.

doi:10.1111/j.1365-2958.2007.05896.x

Cited by 47 publications

(64 citation statements)

References 63 publications

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“…Thus, SpoVE localizes in a pattern that closely matches the membrane dynamics of the engulfing forespores. Further, the pattern at later time points (post-3 h) is consistent with the role of SpoVE in spore cortex synthesis, a late (postengulfment) event in sporulation (50). The examination of SpoVE-YFP expression by immunoblotting analysis of samples taken at equivalent time points demonstrated that the protein appeared at 3 h, consistent with the results of microscopy (data not shown).…”

Section: Localization Of Spovesupporting

confidence: 75%

“…spoVE, which during sporulation is expressed under the control of the mother cell-specific transcription factor E (45), is not essential for vegetative growth, yet it is absolutely required for the production of cortex PG. Thus, during sporulation, ⌬spoVE mutants fail to form a cortex (15,34,50) and they accumulate cytoplasmic PG precursors (50), suggesting a defect at an early step in PG polymerization and consistent with the view that the SEDS proteins may function in lipid-linked precursor translocation. SpoVE may be part of a third putative PG biosynthetic complex of B. subtilis and other endospore-forming bacteria which includes a sporulation-specific, E -controlled PBP, called SpoVD, required for synthesis of the cortex (4).…”

supporting

confidence: 54%

“…Like all other genes known to be required for spore cortex formation (50), spoVE is under the control of E , a mother cell-specific transcription factor (45). Therefore, SpoVE is likely to be found at the outer forespore membrane, surrounding the developing spore, and facing the mother cell cytoplasm.…”

Section: Localization Of Spovementioning

confidence: 99%

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Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein

et al. 2008

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The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS ("shape, elongation, division, and sporulation") family of proteins.Bacterial shape is determined by an extracellular structure composed of peptidoglycan (PG), a rigid polymer of repeated subunits of a disaccharide peptide monomer. This giant macromolecule is found on the outside of the cytoplasmic membrane of nearly all eubacteria. A series of essential and highly conserved enzymes convert UDP-GlcNAc to lipid II, the UDPmuramyl pentapeptide (47). To form mature PG, lipid II is translocated across the cytoplasmic membrane via an uncharacterized mechanism and is added to the preexisting cell wall through transpeptidation and transglycosylation reactions mediated by members of the PBP (penicillin binding protein) family of proteins. Little is known about the mechanism of lipid II transport across the cytoplasmic membrane other than that it is probably dependent on a protein(s) since it does not occur spontaneously across lipid bilayers (46).The integral membrane proteins RodA and FtsW are members of what has been termed the SEDS ("shape, elongation, division, and sporulation") family of integral membrane proteins (16, 18) that are present in all cell wall-containing bacteria. Several lines of evidence are consistent with the participation of RodA and FtsW in the translocation of lipid II during cell elongation and cell division, respectively (17, 19), although this hypothesis has not been subjected to a direct test. For instance, depletion of RodA leads to a block in lateral cell growth (16), although RodA is not strictly essential for cell viability. Null mutants exhibit slow growth and small cell diameters and are viable in minimal medium (7). Also, temperature-sensitive Escheri...

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Section: Localization Of Spovesupporting

confidence: 75%

supporting

confidence: 54%

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Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein

et al. 2008

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“…1; Table 2). Consistent with the proposed role of MviN as the lipid II flippase, strains lacking SpoVB are defective in the synthesis of spore cortex peptidoglycan and accumulate peptidoglycan precursors (32).…”

Section: Resultsmentioning

confidence: 70%

Bacillus subtilisHomologs of MviN (MurJ), the PutativeEscherichia coliLipid II Flippase, Are Not Essential for Growth

Fay

Dworkin

2009

J Bacteriol

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Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a ⌬spoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.Peptidoglycan synthesis is vital for cell growth and maintenance of cell shape in both gram-positive and gram-negative bacteria. This polymer of glycan chains that are cross-linked by peptide bridges forms an extracellular shell which provides protection against osmotic stresses as well as a sturdy scaffolding for extracellular appendages. The enzymes responsible for peptidoglycan synthesis are highly conserved in all bacteria with a cell wall. In the cytoplasm, the enzymes MurA to MurE synthesize the soluble MurNAc-pentapeptide starting with UDP-GlcNAc. MraY links this molecule to an isoprenoid chain, forming the membrane-associated lipid I precursor. MurG then adds UDP-GlcNAc to make lipid II, which is subsequently flipped across the cytoplasmic membrane and attached by penicillin-binding proteins via transglycosylation and transpeptidation reactions to the mature peptidoglycan.While these cytoplasmic and extracellular steps are well characterized, comparatively little is known about the mechanism of membrane translocation. Fluorescently tagged lipid II does not spontaneously flip in protein-free liposomes (31), as would be expected given its large hydrophilic carbohydrate and protein groups. This observation suggests that that flipping is a protein-mediated process, and, consistent with this prediction, fluorescent lipid II molecules were translocated across vesicles made from Escherichia coli membranes. Genetic data have pointed to proteins belonging to the SEDS family as potential lipid II flippases (14). These proteins are highly conserved and contain multiple membrane-spanning domains (generally 10 to 12 transmembrane helices). Since they are in most cases essential for viability, it has been problematic to demonstrate their function. However, depletion or temperature-sensitive mutations result in phenotypes consistent with a block in peptidoglycan synthesis. A nonessential SEDS protein, Bacillus subtilis SpoVE, is necessary for the formation of peptidoglycan during a later step in spore development (13), and point mutations in SpoVE block peptidoglycan synthes...

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“…These proteins have also been shown to mediate resistance to a wide range of cationic dyes, fluoroquinolones, aminoglycosides, and other structurally diverse antibodies and drugs (15). MATE proteins have also been implicated in the production of polysaccharides, such as RfbX (Wzx) (4) and WzxE (17), which have been implicated in E. coli Oantigen biosynthesis; Bacillus subtilis SpoVB, which is involved in spore cortex biosynthesis (16,19); and eukaryotic RFT1, which is required for translocation of the dolichyl diphosphateMan 5 GlcNAc 2 intermediate, an oligosaccharide complex used in protein glycosylation, from the cytosolic side of the endoplasmic reticulum membrane to the lumen during the biosynthesis of dolichyl diphosphate-Glc 3 Man 9 GlcNAc 2 (8). These results suggest that MviN may be involved in the transmembrane transport of peptidoglycan precursors across the inner membrane.…”

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confidence: 99%

Involvement of an Essential Gene,mviN, in Murein Synthesis inEscherichia coli

et al. 2008

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We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.The mviN gene was first identified in Salmonella enterica serovar Typhimurium as a gene in a chromosomal region required for virulence in a mouse model of typhoidlike disease (1, 3). One of the putative virulence genes in this region was designated mviS (mouse virulence Salmonella). Subsequently, mviS was identified as fliA, a gene that encodes a sigma factor necessary for expression of the flg operon, which includes 14 genes that are essential for flagellum synthesis (2,3,5,14). Thus, although the original terminology has been maintained, there is no direct evidence that mviN is involved in virulence, and its function has not been fully elucidated.In our lab, we are carrying out systematic construction of Escherichia coli long-range chromosomal deletion mutants (7,12). Previously, we reported that the chromosomal region containing mviN is essential for cell growth. Viable mutants having a deletion of this chromosomal region were isolated only in the presence of a mini-F plasmid carrying the rimJ-yceH-mviMmviN locus (12). To determine whether mviM and mviN are

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Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K

Cited by 47 publications

References 63 publications

Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein

Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein

Bacillus subtilisHomologs of MviN (MurJ), the PutativeEscherichia coliLipid II Flippase, Are Not Essential for Growth

Involvement of an Essential Gene,mviN, in Murein Synthesis inEscherichia coli

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