Cell Wall Protein in Bacillus subtilis

The cell wall of Bacillus subtilis 168 contains protein that is refractory to removal by salts, detergents, and denaturants.     

Cell Wall and Morphological Changes Induced by Temperature Shift in Bacillus subtilis Cell Wall Mutants

Bacillus subtilis RUB1012 and RUB1013 have the following phenotype when grown at 45 degrees C: no growth on tryptose blood agar base, growth as clumps of spheres in broth culture, a slow autolysis rate, and a low proportion of teichoic acid to peptidoglycan. Revertants of strain RUB1012 (RUB2032, RUB2012, and RUB2042) that could grow on tryptose blood agar base were isolated. Each revertant had a different proportion of teichoic acid to peptidoglycan. The nanomoles of phosphorus per milligram of cell wall at the nonpermissive temperature were 141, 160, 236, and 541 for strain RUB1012 and revertants RUB2032, 2012, and 2042, respectively, as compared with 1,100 for the parent strain. With most bacteriophage tested, plating efficiency was related to the amount of glucosylated teichoic acid. Scanning electron microscopy was used to study strain RUB2032 during a shift from growth at 30 degrees C to growth at 45 degrees C. The change from rod to sphere began with the thickening of the cylindrical portion of the cell. Caps of the cells appeared to be immune to the thickening process. During growth, the cells became progressively shorter and thicker, and cell separation was inhibited. When cells of strain RUB2032 were shifted from growth at 45 degrees C to growth at 30 degrees C, accumulation of an amorphous material on the outer surfaces of the cells preceded the change from sphere to rod morphology. Cells remained clumped, with rods appearing at the periphery of the clumps. Analysis by DNA-mediated transformation and PBS1-mediated transduction indicated that strains RUB1012 and RUB1013 have multiple mutations mapping in the same region as other cell wall mutations.     

Cell Wall Turnover at the Hemispherical Caps of Bacillus subtilis

Cell walls made by Bacillus subtilis bacteria grown in D(2)O medium have buoyant densities in CsCl which are different from walls made by cells grown in H(2)O medium. Cell wall turnover was studied by measuring the change in wall buoyant density after a B. subtilis culture was shifted from growth in D(2)O medium to aeration in H(2)O medium. Walls from the hemispherical caps were isolated after preferential digestion of wall from the cylindrical regions using the B. subtilis autolytic amidase. The walls from the polar regions were found to turn over extensively.     

Interaction of Concanavalin A with the Cell Wall of Bacillus subtilis

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.     

The lytE Gene of Bacillus subtilis 168 Encodes a Cell Wall Hydrolase

Bacillus subtilis cell wall-bound protein CWBP33 is encoded by lytE, a gene expressed during the exponential growth phase. Sequence analysis of LytE, a 33-kDa protein, reveals two domains. The N-terminal domain contains a threefold-repeated motif common to several peptidoglycan binding proteins, while the C-terminal domain, probably carrying the catalytic activity, has homology with certain exoproteins. Zymographs unambiguously reveal that the absence of CWBP33, due to inactivation of lytE, is accompanied by the loss of a lytic activity. In lytE mutants, the cell autolysis rate is significantly decreased, although autolysis of corresponding, purified cell walls does not seem to be affected.     

Engulfment during sporulation in Bacillus subtilis is governed by a multi-protein complex containing tandemly acting autolysins

The conversion of a growing cell into an endospore in Bacillus subtilis involves a phagocytic-like process in which the developing spore (the forespore) is wholly engulfed by the adjacent mother cell. A prerequisite for engulfment is the removal of peptidoglycan from the septum that separates the forespore from the mother cell, a process that depends on the autolysin SpoIID and two proteins of unknown function, SpoIIM and SpoIIP. Here we present evidence that SpoIIP is also an autolysin, that it acts in tandem with SpoIID, and that all three proteins are in a complex with each other. We further show that the members of the complex exhibit a hierarchical relationship in which SpoIIM is responsible for localization to the septal membrane, SpoIIP localizes to the septal membrane by interacting with SpoIIM, and SpoIID, in turn, localizes by interacting with SpoIIP. Finally, we show that localization of SpoIIM depends on a fourth protein SpoIIB, raising the possibility that the complex contains an additional component and creating an overall hierarchy of the form: SpoIIB-->SpoIIM-->SpoIIP-->SpoIID.     

Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.The Bacillus subtilis spoVB gene was first identified as a locus in which a mutation could produce a block at a late stage of spore development (14, 30). Analysis of this locus revealed that it encoded an apparent integral membrane protein (33), and a detailed analysis of a spoVB null mutant demonstrated a block at a very early step in synthesis of the spore cortex peptidoglycan (PG) (40). The mutant synthesized essentially no cortex and accumulated cytoplasmic PG precursors, the same phenotype found in other mutant strains blocked in functions known to be directly involved in PG polymerization (40). These results suggested that SpoVB plays a direct role in assembly or function of the spore PG synthesis apparatus.PG synthesis is a highly conserved and complex process that must span the cell membrane (reviewed in reference 38). Soluble nucleotide-linked PG precursors are synthesized in the cytoplasm. N-Acetylmuramic acid with a pentapeptide side chain is then transferred to an undecaprenol lipid carrier to produce lipid I, with subsequent addition of N-acetylglucosamine to produce lipid II, undecaprenyl-pyrophosphoryl-N-acetylmuramic acid (pentapeptide)-N-acetylglucosamine. Lipid II is then flipped across the membrane via an unknown mechanism. Two families of proteins have been postulated to perform this function: the SEDS family of integral membrane proteins, including FtsW, RodA, and SpoVE (13), and, more recently, the COG2244 family (23), which includes SpoVB and the MviN (MurJ) protein of Escherichia coli (35). In both cases, loss of a protein within one of these families has been shown to result in a block in PG synthesis and the accumulation of lipid-linked and/or soluble PG precursors (16, 20, 35, 40).In the standard model of PG synthesis, flippase activity brings the disaccharide-pentapeptide moieties to the penicillin-binding proteins (PBPs), which polymerize the PG macromolecule on the outer surface of the membrane (39). The class A, high-molecular-weight PBPs possess an N-terminal glycosyl transferase domain that polymerizes the disaccharides into polysaccharide chains (38). These chains are cross-linked via the transpeptidase activity within the penicillin-binding, C-terminal domains of both the class A and the class B PBPs. The N-terminal domains of the class A PBPs and the closely related monofunctional glycosyl transferases found in some species are the only defined PG glycan strand polymerases, and in several species the presence of at least one of these enzymes is essential. However, in B. subtilis (26) and Enterococcus faecalis (3), strains lacking all of these known glycosyl transferases are viable and produce PG walls, indicating the presence of another glycosyl transferase capable of this activity. This alternate glycosyl transferase is distinct in that it is relatively resistant to moenomycin (3, 26), an inhibitor of the class A PBP glycosyl transferase activity (6).Given the strong and early block in cortex PG polymerization observed to occur in a spoVB mutant (40), we wished to further analyze the potential role of this class of protein. SpoVB is a member of a relatively large family of proteins, COG2244 (23), some of which are involved in polymerization of other polysaccharides in bacteria, archaea, and eukaryotes. Bioinformatic analysis has generally predicted that these proteins span the membrane 12 to 14 times, and in some cases experimental evidence has supported this structure (7, 24). A role generally ascribed to these proteins is the flipping of lipid-linked oligosaccharides, produced on the inner face of a membrane, to the outside, where the oligosaccharides are then further polymerized or transferred to other substrates. Some prominent members of this family include Wzx, which functions in O-antigen synthesis in gram-negative bacteria (41); TuaB, which functions in teichuronic acid synthesis in B. subtilis (36); and Rft1, which functions in protein glycosylation in eukaryotes (12). MviN is essential in some gram-negative species, including Burkholderia pseudomallei, E. coli, and Sinorhizobium meliloti (22, 34), and has been shown to play a role in E. coli PG synthesis (16, 35). A Rhizobium tropici mutation that truncates mviN approximately 50% into the coding sequence was not lethal (29). However, it is not known whether this was the sole mviN homolog in the genome or whether the truncated gene product might be functional.We have analyzed the phenotypic properties of B. subtilis strains lacking other proteins within the COG2244 family that are most closely related to SpoVB. Results suggest that these proteins also play roles in PG synthesis and that, in one case, this role is in a synthetic system that is relatively moenomycin resistant. We postulate that these proteins function in an alternate pathway for PG synthesis that may involve the flipping of lipid-linked PG oligosaccharides rather than lipid II disaccharides.     

Mechanism of Anucleate Cell Production in the oriC-Deleted Mutants of Bacillus subtilis

We constructed oriC-deleted mutants of Bacillus subtilis byintegrating the minimal replication region of plasmid pLS32into the proA (115°), spoIIIJ (360°) and thrS (256°)loci of the chromosome, respectively. All three mutants producedanucleate cells and the DNA/protein ratio was lower than thatof the wild-type strain when grown in nutrient broth. However,when grown in minimal-glucose medium, the frequency of anucleatecells was reduced in all of them and the DNA/protein ratio wasrestored to normal. Especially, the oriC-deleted mutant in whichthe plasmid was integrated near oriC produced almost no anucleatecell. These results indicate that initiation frequency of chromosomereplication from the integrated plasmid origin were reduceddisproportionately to cell mass increase in rich medium, whichin turn disrupted coordination between DNA replication cycleand cell division cycle. The locations of the plasmid originrelative to the natural oriC locus affected the production ofanucleate cell remarkably, suggesting that partition mechanismof chromosome was also impaired by the translocation of itsreplication origin.     

Mechanism of uridine production by Bacillus subtilis mutants

Summary Pyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.Microbial production of uridine. Part III     

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

An Interpretation of Several Cell Wall Stains Applied to Heat-Fixed Bacillus subtilis On Glass Slides

The Dyer, Chance, alcian blue, and tannic acid-crystal violet wall stains for bacteria were compared. All gave apparent cell wall widths which were considerably greater than those demonstrated by electron microscope methods. The exaggerated width, as seen by the light microscope, was apparently due to staining of a portion of the cytoplasmic material adjacent to the wall, and in addition, to a precipitate on the surface of the cell produced by the Dyer and the Chance stains. Heat-fixed cells on slides were shown to retain considerable height or third dimension, being about half as high as wide. Moreover, such cells had their walls flattened against the slide to form a collar-like projection around the periphery of the shrunken protoplasm. This latter effect alone was not sufficient to explain the exaggerated wall thickness shown by staining. For obtaining reliable measurements of cell widths, the nigrosin negative stain was found to be as good as any, provided that the thickness of its film were controlled. Negative stains were used so that comparisons of cell widths shown by them and by positive stains could be made. This, in turn, facilitated the detection of the apparent widening of cell bodies caused by dye precipitates on their surfaces.