Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus.

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

The outer membrane is an essential load-bearing element in Gram-negative bacteria.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis.

The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis.

Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway.

Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with ß-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

Variations in the binding pocket of an inhibitor of the bacterial division protein FtsZ across genotypes and species.

The recent increase in antibiotic resistance in pathogenic bacteria calls for new approaches to drug-target selection and drug development. Targeting the mechanisms of action of proteins involved in bacterial cell division bypasses problems associated with increasingly ineffective variants of older antibiotics; to this end, the essential bacterial cytoskeletal protein FtsZ is a promising target. Recent work on its allosteric inhibitor, PC190723, revealed in vitro activity on Staphylococcus aureus FtsZ and in vivo antimicrobial activities. However, the mechanism of drug action and its effect on FtsZ in other bacterial species are unclear. Here, we examine the structural environment of the PC190723 binding pocket using PocketFEATURE, a statistical method that scores the similarity between pairs of small-molecule binding sites based on 3D structure information about the local microenvironment, and molecular dynamics (MD) simulations. We observed that species and nucleotide-binding state have significant impacts on the structural properties of the binding site, with substantially disparate microenvironments for bacterial species not from the Staphylococcus genus. Based on PocketFEATURE analysis of MD simulations of S. aureus FtsZ bound to GTP or with mutations that are known to confer PC190723 resistance, we predict that PC190723 strongly prefers to bind Staphylococcus FtsZ in the nucleotide-bound state. Furthermore, MD simulations of an FtsZ dimer indicated that polymerization may enhance PC190723 binding. Taken together, our results demonstrate that a drug-binding pocket can vary significantly across species, genetic perturbations, and in different polymerization states, yielding important information for the further development of FtsZ inhibitors.