Stéphane Mesnage

Bacterial SLH domain proteins are non‐covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non‐covalently anchored to the cell surface via an S‐layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non‐covalent interaction between the SLH domain and peptidoglycan‐associated polymers. Here we report the characterization of a two‐gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH‐containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan‐associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH‐containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S‐layer proteins and the Bacillus subtilis levansucrase

Many surface proteins of Gram‐positive bacteria contain motifs, about 50 amino acids long, called S‐layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S‐layer proteins, each with three SLH motifs towards the amino‐terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three‐motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitrodemonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three‐motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface‐exposed levansucrase retained its enzymatic and antigenic properties.

Developmental switch of S-layer protein synthesis in Bacillus anthracis

Adjustment of the synthesis of abundant protein to the requirements of the cell involves processes critical to the minimization of energy expenditure. The regulation of S‐layer genes might be a good model for such processes because expression must be controlled, such that the encoded proteins exactly cover the surface of the bacterium. Bacillus anthracis has two S‐layer genes, sap and eag, encoding the S‐layer proteins Sap and EA1 respectively. We report that the production and surface localization of Sap and EA1 are under developmental control, suggesting that an exponential phase ‘Sap layer’ is subsequently replaced by a stationary phase ‘EA1 layer’. This switch is controlled at the transcriptional level: sap is most certainly transcribed by RNA polymerase containing σA, whereas eag expression depends on σH. More importantly, Sap is required for the temporal control of eag, and EA1 is involved in strict feedback regulation of eag. This control may be direct because both S‐layer proteins bind, in vitro, the eag promoter, specifically suggesting that they might act as transcriptional repressors.

Super‐resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super‐resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super‐resolution three‐dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis.

Bacillus anthracis synthesizes two S‐layer proteins, each containing three S‐layer homology (SLH) motifs towards their amino‐terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface‐exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.

Molecular basis for bacterial peptidoglycan recognition by LysM domains.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM–peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

Functional Analysis of AtlA, the Major N-Acetylglucosaminidase of Enterococcus faecalis

The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

Bacillus anthracis surface: capsule and S-layer

Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S‐layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three ‘S‐layer homology’ motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co‐localized at the cell surface of both the non‐capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S‐layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a Gram‐positive bacterium.

Role of N-Acetylglucosaminidase and N-Acetylmuramidase Activities in Enterococcus faecalis Peptidoglycan Metabolism

Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[14C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

Bacillus anthracis Cell Envelope Components

Bacillus anthracis is a Gram-positive bacterium harboring a complex parietal architecture. The cytoplasmic membrane is surrounded by a thick peptidoglycan of the A1 gamma type. Only one associated polymer, a polysaccharide composed of galactose, N-acetylglucosamine, and N-acetylmannosamine, is covalently linked to the peptidoglycan. Outside the cell wall is an S-layer. Two proteins can each compose the S-layer. They are noncovalently anchored to the cell wall polysaccharide by their SLH N-terminal domain. The poly-gamma-D-glutamate capsule, which covers the S-layer, has an antiphagocytic role and its synthesis is dependent on environmental factors mimicking the mammalian host, such as bicarbonate and a temperature of 37 degrees C.