A role for the Gram-negative outer membrane in bacterial shape determination.

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod complex (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod complex. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria.

A role for the Gram-negative outer membrane in bacterial shape determination.

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod system (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod system. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria. SIGNIFICANCE: The cell wall has traditionally been thought to be the main structural determinant of the bacterial cell envelope that resists internal turgor and determines cell shape. However, the outer membrane (OM) has recently been shown to contribute to the mechanical strength of Gram-negative bacterial envelopes. Here, we demonstrate that changes to OM composition predicted to increase its load bearing capacity rescue the growth and shape defects of Escherichia coli mutants defective in the major cell wall synthesis machinery that determines rod shape. Our results therefore reveal a previously unappreciated role for the OM in bacterial shape determination in addition to its well-known function as a diffusion barrier that protects Gram-negative bacteria from external insults like antibiotics.

Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery.

The bacterial peptidoglycan (PG) cell wall maintains cell shape and prevents osmotic lysis. During growth of rod-shaped cells, PG is incorporated along the cell cylinder by the RodA-PBP2 synthase of the multi-protein Rod system (elongasome). Filaments of the actin-like MreB protein orient synthesis of the new PG material. They are connected to the RodA-PBP2 synthase in part through the RodZ component. MreC and MreD are other conserved components of the system, but their function is not well understood. Amino acid changes in RodA-PBP2 were recently identified that bypass a requirement for MreC and MreD function, suggesting the Mre proteins act as activators of the synthase. To further investigate their function, we developed a genetic strategy to identify dominant-negative alleles of mreC and mreD in Escherichia coli Residues essential for Rod system function were identified at the junction of two subdomains within MreC and in a predicted ligand-binding pocket of MreD. Additionally, we found that although the proline-rich C-terminal domain of MreC is non-essential, substitutions within this region disrupt its function. Based on these results, we propose that the C-terminus of MreC and the putative ligand-binding domain of MreD play regulatory roles in controlling Rod system activity.IMPORTANCE: Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multi-protein machine called the Rod system (elongasome) has long been implicated in rod-shape determination in bacilli. However, the functions of many of its conserved components remain unclear. Here, we describe a new genetic system to dissect the function of these proteins and how we used it to identify potential regulatory domains within them that may modulate the function of the shape-determining machinery.

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG-defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain-function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.