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.

The LpoA activator is required to stimulate the peptidoglycan polymerase activity of its cognate cell wall synthase PBP1a.

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.

Breaking on through to the other side: protein export through the bacterial Sec system.

More than one-third of cellular proteomes traffic into and across membranes. Bacteria have invented several sophisticated secretion systems that guide various proteins to extracytoplasmic locations and in some cases inject them directly into hosts. Of these, the Sec system is ubiquitous, essential and by far the best understood. Secretory polypeptides are sorted from cytoplasmic ones initially due to characteristic signal peptides. Then they are targeted to the plasma membrane by chaperones/pilots. The translocase, a dynamic nanomachine, lies at the centre of this process and acts as a protein-conducting channel with a unique property; allowing both forward transfer of secretory proteins but also lateral release into the lipid bilayer with high fidelity and efficiency. This process, tightly orchestrated at the expense of energy, ensures fundamental cell processes such as membrane biogenesis, cell division, motility, nutrient uptake and environmental sensing. In the present review, we examine this fascinating process, summarizing current knowledge on the structure, function and mechanics of the Sec pathway.