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Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth., Competing Interests: Declaration of Competing Interest The authors do not have any conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Contractile injection systems (CISs) are phage tail-like machineries found in a wide range of bacteria. They are often deployed by bacteria to translocate effectors into the extracellular space or into target cells. CISs are classified into intracellular type VI secretion systems (T6SSs) and extracellular CIS (eCISs). eCISs are assembled in cytoplasm and released into the extracellular milieu upon cell lysis, while T6SSs are the secretion systems widespread among Gram-negative bacteria and actively translocate effectors into the environment or into the adjacent cell, without lysis of T6SS-producing cells. Recently, several noncanonical CISs that exhibit distinct characteristics have been discovered. This review will provide an overview on these noncanonical CISs and their unique features, as well as new advances in reprogramming CISs for therapeutic protein delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Intravacuolar bacterial pathogens establish intracellular niches by constructing membrane-encompassed compartments. The vacuoles surrounding the bacteria are remarkably stable, facilitating microbial replication and preventing exposure to host cytoplasmically localized innate immune sensing mechanisms. To maintain integrity of the membrane compartment, the pathogen is armed with defensive weapons that prevent loss of vacuole integrity and potential exposure to host innate signaling. In some cases, the microbial components that maintain vacuolar integrity have been identified, but the basis for why the compartment degrades in their absence is unclear. In this review, we point out that lessons from the microbial-programmed degradation of the vacuole by the cytoplasmically localized Shigella flexneri provide crucial insights into how degradation of pathogen vacuoles occurs. We propose that in the absence of bacterial-encoded guard proteins, aberrant trafficking of host membrane-associated components results in a dysfunctional pathogen compartment. As a consequence, the vacuole is poisoned and replication is terminated., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recent studies show that internal organization of bacterial cells is more complex than previously appreciated. A clear example of this is the assembly of the nanoscale membrane platforms termed functional membrane microdomains. The lipid composition of these regions differs from that of the surrounding membrane; these domains confine a set of proteins involved in specific cellular processes such as protease secretion and signal transduction. It is currently thought that functional membrane microdomains act as oligomerization platforms and promote efficient oligomerization of interacting protein partners in bacterial membranes. In this review, we highlight the most noteworthy achievements, challenges and controversies of this emerging research field over the past five years., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Gram-negative rod-shaped bacteria have different approaches to position the cell division initiating Z-ring at the correct moment in their cell division cycle. The subsequent maturation into a functional division machine occurs in vastly different species in two steps with appreciable time in between these. The function of this time delay is unclear, but may partly be explained by competition for Lipid-II between proteins involved in length growth that interact directly with the Z-ring early in the maturation phase and the proteins involved in septum synthesis. A second possible activity of the early Z-ring might be the monitoring of or the active involvement in DNA segregation through proteins such as ZapA and ZapB/MatP and their homologues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The divisome and elongasome are bacterial protein complexes responsible for peptidoglycan (PG) synthesis during cell division and elongation, respectively. We review several lines of evidence, arguing for a shared evolutionary past of the divisome and elongasome. Both integrate closely related penicillin-binding proteins (PBPs) for PG synthesis, use proteins of the RodA/FtsW (SEDS, shape, elongation, division and sporulation) family for Lipid II export and interact with MraY/Mur proteins for Lipid II synthesis. It was recently shown that the actin-like protein FtsA of the divisome polymerises on membranes, adding another parallel, since membrane-associated filaments of the bacterial actin MreB guide the elongasome. Given these similarities, it seems plausible to conclude that the elongasome is a modified version of the divisome, without the membrane-constricting FtsZ-ring and its associated machinery on the inside., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide is a highly acylated saccharolipid located on the outer leaflet of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is critical to maintaining the barrier function preventing the passive diffusion of hydrophobic solutes such as antibiotics and detergents into the cell. Lipopolysaccharide has been considered an essential component for outer membrane biogenesis and cell viability based on pioneering studies in the model Gram-negative organisms Escherichia coli and Salmonella. With the isolation of lipopolysaccharide-null mutants in Neisseria meningitidis, Moraxella catarrhalis, and most recently in Acinetobacter baumannii, it has become increasingly apparent that lipopolysaccharide is not an essential outer membrane building block in all organisms. We suggest the accumulation of toxic intermediates, misassembly of essential outer membrane porins, and outer membrane stress response pathways that are activated by mislocalized lipopolysaccharide may collectively contribute to the observed strain-dependent essentiality of lipopolysaccharide., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bacteria express a multitude of hair-like adhesive appendages on their cell surfaces, together referred to as pili or fimbriae. In Gram-negative bacteria, these proteinaceous structures are assembled through a number of dedicated secretion pathways including the chaperone-usher pathway, the nucleation/precipitation pathway and the type IV pilus pathway. Pili are prevalent in pathogenic strains and play important roles in the establishment and persistence of bacterial infections by mediating host cell adhesion, cell invasion or biofilm formation. Their indispensible roles in pathogenesis render them attractive targets for directed therapeutic intervention. Here, we describe the recent advances in the chemical attenuation of pilus-associated virulence in Gram-negative bacteria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The penicillin-binding proteins (PBPs) polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. As part of this process, the PBPs help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Genetic and microscopic studies reveal clear morphological responsibilities for class A and class B PBPs and suggest that the mechanism of shape determination involves differential protein localization and interactions with specific cell components. In addition, the low molecular weight PBPs, by varying the substrates on which other PBPs act, alter peptidoglycan synthesis or turnover, with profound effects on morphology.

Research on the molecular mechanism of bacterial flagellar assembly has been an ongoing study that spans three decades. Early work showed that regulation of flagellar gene transcription was coupled to the assembly process. Recent advances in the understanding of the regulation of flagellar assembly have shown that translational and post-translational regulation also plays a significant role in flagellar assembly. In both Salmonella and Caulobacter crescentus, translational regulation influences the secretion of the anti-sigma(28) factor FlgM and the flagellin fljK, respectively. Post-translational regulatory mechanisms also control the length of the hook and the ability of the type III secretion system to discriminate between middle and late secretion substrates. The flagellum provides a model system for understanding how gene regulation functions to ensure the efficient assembly of a complex structure and fundamental mechanisms common to all type III secretion systems.

Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules. Their reactions to these changes require a series of rapid adaptive responses. Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments. Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents. In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm. Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure. Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

Swarming involves differentiation of vegetative cells into hyperflagellated swarm cells that undergo rapid and coordinated population migration across solid surfaces. Cell density, surface contact, and physiological signals all provide critical stimuli, and close cell alignment and the production of secreted migration factors facilitate mass translocation. Flagella biogenesis is central to swarming, and the flhDC flagellar master operon is the focal point of a regulatory network governing differentiation and migration.

Current models of both heat induction and the chaperone-mediated feedback control of the sigma32 regulon in Escherichia coli have been further substantiated, and the extent of conservation among Gram-negative bacteria has been assessed. Analyses of the 'CIRCE' and other regulons or operons in Gram-positive and Gram-negative bacteria have provided new insights into their significance and regulatory mechanisms.

Many Gram-negative bacteria that cause disease in either mammals or plants share a strategy of delivering toxic proteins into the cytoplasm of host cells known as type III secretion. Recent advances have provided a glimpse at the molecular nature of these lethal injection machines. Several groups have reported fibrous structures on bacterial surfaces that appear to be extensions of type III machines and necessary for toxin injection into host cells. Other research revealed complex mechanisms of secretion substrate recognition that presumably function to direct toxins to different locations during infection.

Multiple antibiotic resistance in bacteria was at first thought to be caused exclusively by the combination of several resistance genes, each coding for resistance to a single drug. More recently, it became clear that such phenotypes are often achieved by the activity of drug efflux pumps. Some of these efflux pumps exhibit an extremely wide specificity covering practically all antibiotics, chemotherapeutic agents, detergents, dyes, and other inhibitors, the exception perhaps being very hydrophilic compounds. Such efflux pumps work with exceptional efficiency in Gram-negative bacteria through their synergistic interaction with the outer membrane barrier. It is disturbing that the antibacterial agents of the most advanced type, which are unaffected by common resistance mechanisms, are precisely the compounds whose use appears to select for multidrug-resistant mutants that overproduce these efflux pumps of wide specificity.

A variety of Gram-negative bacteria produce membrane permeant, acylated homoserine lactone (HL) pheromones that act as cell density cues. Synthesis and response to these factors requires proteins homologous to the Luxl acylhomoserine lactone synthase and the LuxR transcription factor from Vibrio fischeri. Recent genetic and biochemical studies have begun to provide a mechanistic understanding of acyl HL dependent gene regulation. Examination of the role of acyl HLs in diverse bacteria positions LuxR-Luxl type systems within an increasingly broad regulatory context and suggests that, in some bacteria, they comprise a global regulatory circuit.