SummaryBacterial spores are surrounded by a morphologically complex, mechanically flexible protein coat, which protects the spore from toxic molecules. The interactions among the over 50 proteins that make up the coat remain poorly understood. We have used cell biological and protein biochemical approaches to identify novel coat proteins in Bacillus subtilis and describe the network of their interactions, in order to understand coat assembly and the molecular basis of its protective functions and mechanical properties. Our analysis characterizes the interactions between 32 coat proteins. This detailed view reveals a complex interaction network. A key feature of the network is the importance of a small subset of proteins that direct the assembly of most of the coat. From an analysis of the network topology, we propose a model in which low-affinity interactions are abundant in the coat and account, to a significant degree, for the coat's mechanical properties as well as structural variation between spores.
Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.The structural element of the bacterial cell wall is peptidoglycan (PG), and the importance of this structure is apparent in the number of antimicrobial agents targeting its components and the enzymatic reactions leading to its synthesis. The glycosyltransferase (GT) activity carrying out one of the final enzymatic reactions in PG synthesis is an attractive target, because the enzymes performing that activity are highly conserved and believed to be essential (9, 10). PG is composed of glycan strands cross-linked by peptide side chains. The glycan strands are synthesized by a GT that adds lipid-linked disaccharide pentapeptide subunits to nascent glycan strands. The pentapeptides are then utilized by a transpeptidase to crosslink adjacent glycan strands (reviewed in reference 4). Most of the proteins involved in these final enzymatic reactions are penicillin-binding proteins (PBPs), which are divided into three classes based on the presence of conserved functional domains (9, 10). Only the class A PBPs have an N-terminal domain that contains the conserved amino acid sequences found in all GTs clearly demonstrated to polymerize PG. Class B PBPs have a different N-terminal domain; and although some researchers have associated GT activity with class B PBPs (15), others have been unable to reproduce those results (2). Both classes have C-terminal penicillin-binding domains containing the transpeptidase activity that cross-links peptide side chains. Some species also have monofunctional glycosyltransferases (MGTs) that contain the conserved amino acid sequences found in the class A PBP GT domain but whic...
Aims: To develop test methods and evaluate the survival of Bacillus anthracis ΔSterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. Methods and Results: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6Á5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. Conclusions: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ΔSterne and B. thuringiensis Al Hakam spores with similar kinetics. Significance and Impact of the Study: Hot, humid air is a potential alternative to conventional chemical decontamination.
The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.
The four class A penicillin-binding proteins (PBPs) of Bacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamed pbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic ␦-lactam and L-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.Peptidoglycan (PG) is the essential structural element that provides shape and stability to most bacterial cells. In the dormant endospore PG is required for the maintenance of spore core dehydration and therefore for spore heat resistance. Both vegetative-cell and spore PGs are composed of glycan strands of repeating N-acetylglucosamine and N-acetyl muramic acid residues cross-linked by peptide side chains (reviewed in reference 2). Polymerization of PG involves the addition of disaccharide pentapeptide subunits onto a growing glycan strand by a glycosyltransferase. The peptide side chains are then utilized by a transpeptidase to cross-link the glycan strands. Side chains that are not utilized for cross-linking are cleaved to tripeptides or tetrapeptides.Spore PG consists of two layers that can be distinguished structurally and functionally. The germ cell wall is adjacent to the inner forespore membrane and serves as the initial cell wall during spore germination and outgrowth. It is surrounded by the cortex, which comprises the outer 70 to 90% of the spore PG (28) and which is rapidly degraded during spore germination (4, 14). Cortex PG is more loosely cross-linked than vegetative PG, and 50% of the N-acetyl muramic acid residues hav...
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