Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation.

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by 'autolysins', enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low-salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

Role of penicillin-binding protein 1b in competitive stationary-phase survival of Escherichia coli.

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis.

Peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. Peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the beta-lactams, imipenems, cephalosporins, and glycopeptides. Resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. Representative structures have been reported for most of the enzymes in the pathway. Until now, however, there have been no structures of any peptidoglycan glycosyltransferases (also known as transglycosylases), which catalyze formation of the carbohydrate chains of peptidoglycan from disaccharide subunits on the bacterial cell surface. We report here the 2.1-A crystal structure of the peptidoglycan glycosyltransferase (PGT) domain of Aquifex aeolicus PBP1A. The structure has a different fold from all other glycosyltransferase structures reported to date, but it bears some resemblance to lambda-lysozyme, an enzyme that degrades the carbohydrate chains of peptidoglycan. An analysis of the structure, combined with biochemical information showing that these enzymes are processive, suggests a model for glycan chain polymerization.

Characterization of IsaA and SceD, two putative lytic transglycosylases of Staphylococcus aureus.

Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

Premature targeting of cell division proteins to midcell reveals hierarchies of protein interactions involved in divisome assembly.

In order to divide, the bacterium Escherichia coli must assemble a set of at least 10 essential proteins at the nascent division site. These proteins localize to midcell according to a linear hierarchy, suggesting that cell division proteins are added to the nascent divisome in strict sequence. We previously described a method, 'premature targeting', which allows us to target a protein directly to the division site independently of other cell division proteins normally required for its localization at midcell. By systematically applying this method to probe the recruitment of and associations among late cell division proteins, we show that this linear assembly model is likely incorrect. Rather, we find that the assembly of most of the late proteins can occur independently of 'upstream' proteins. Further, most late proteins, when prematurely targeted to midcell, can back-recruit upstream proteins in the reverse of the predicted pathway. Together these observations indicate that the late proteins, with the notable exception of the last protein in the pathway, FtsN, are associated in a hierarchical set of protein complexes. Based on these observations we present a revised model for assembly of the E. coli division apparatus.

Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis.

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo.

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.