Outer Membrane Composition Research Articles

The case of the mycomembrane: Investigating the composition and properties of the mycobacterial outer membrane

The case of the mycomembrane: Investigating the composition and properties of the mycobacterial outer membrane

A Robust Protocol to Isolate Outer Membrane Vesicles from Nontypeable Haemophilus influenzae

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition.

Linking the outer membrane composition of salmonella to protective antibody responses.

Invasive non-typhoidal Salmonella (iNTS) infection has a high risk of fatality. Currently, there is no licensed vaccine against iNTS, making creating an effective vaccine priority for regions where the rate of these infections is high. In particular, an ideal vaccine should be protective against multiple serovars of Salmonella. The correlation of the protective properties of antibodies raised against Salmonella with distinct types and lengths of O-antigen repeats and membrane proteins has not been thoroughly investigated yet, but experiments conducted on mice have revealed some important differences. We hypothesize that these differences can be explained by the physical structure of the bacterial outer envelope and the accessibility of proteinaceous epitopes to antibodies. To test this hypothesis, we modeled the outer membrane for two of the most common iNTS serovars, Salmonella enterica Enteritidis (SEn) and Typhimurium (STm), along with the outer membrane proteins OmpD and OmpA. We set up all-atomic molecular simulations (MD) for both proteins separately for the serotypes STm and SEn, and for LPS with different lengths of O4 and O9 O-Ag repeats, giving, overall, 20 systems for 1.5 microseconds each. From these MD simulations, we measured various quantities, including Solvent Accessible Surface Area (SASA) of extracellular loops of the proteins, root-mean-square fluctuation (RMSF) of the residues, and the protein to O-Ag contact area, in order to explain the molecular underpinnings of differential antibody responses being raised by distinct serotypes.

Plasticity and Stereotypic Rewiring of the Transcriptome Upon Bacterial Evolution of Antibiotic Resistance.

Bacterial evolution of antibiotic resistance frequently has deleterious side effects on microbial growth, virulence, and susceptibility to other antimicrobial agents. However, it is unclear how these trade-offs could be utilized for manipulating antibiotic resistance in the clinic, not least because the underlying molecular mechanisms are poorly understood. Using laboratory evolution, we demonstrate that clinically relevant resistance mutations in Escherichia coli constitutively rewire a large fraction of the transcriptome in a repeatable and stereotypic manner. Strikingly, lineages adapted to functionally distinct antibiotics and having no resistance mutations in common show a wide range of parallel gene expression changes that alter oxidative stress response, iron homeostasis, and the composition of the bacterial outer membrane and cell surface. These common physiological alterations are associated with changes in cell morphology and enhanced sensitivity to antimicrobial peptides. Finally, the constitutive transcriptomic changes induced by resistance mutations are largely distinct from those induced by antibiotic stresses in the wild type. This indicates a limited role for genetic assimilation of the induced antibiotic stress response during resistance evolution. Our work suggests that diverse resistance mutations converge on similar global transcriptomic states that shape genetic susceptibility to antimicrobial compounds.

Type III Secretion System Repressor RhpR Induces GrlP, a Glycine-Rich Outer Membrane Lipoprotein with Functions in Regulating the Periplasmic Space and Pleiotropic Responses.

The two-component system RhpRS was initially identified as a regulator of genes encoding the type III secretion system (T3SS) in Pseudomonas syringae. Phosphorylated RhpR (P-RhpR) negatively regulates the T3SS genes by repressing the hrpR promoter, but directly activates the expression of a small gene named here as grlp. Here, we show that grlp is expressed higher in rich medium than in minimal medium in P. s. pv. tomato DC3000 and encodes a glycine rich lipoprotein (GrlP) located in the outer membrane (OM). The grlp gene has a pleiotropic effect on bacterial behaviors such as reductions in pathogenicity, swimming motility, biofilm formation, tolerance to various stresses and antibiotics, and long-term survival when overexpressed, but induces these responses when it is deleted in P. s. pv. tomato DC3000. Overexpression of grlp increases the size of periplasm while deletion of grlp decreases the periplasmic space. Further, GrlP interacts with OprI, the ortholog of E. coli OM lipoprotein Lpp, a key player in determining the size of periplasm and mechanic stiffness of the OM by tethering the OM to peptidoglycan (PG) in periplasm. As periplasmic space and OM mechanics play central roles in regulating bacterial physiology, we speculate that GrlP probably imposes its functions on bacterial physiology by regulating the periplasmic space and OM mechanics. These findings suggest that the T3SS gene regulation is closely coordinated with bacterial cell envelope properties by RhpRS in P. syringe. IMPORTANCE The OM of Gram-negative bacteria is the most front line in contact with extracellular milieu. OM is not only a protective layer, but also a structure that determines the envelope stiffness. Recent evidence indicated that components determining the periplasmic space and cross-links of lipopolysaccharide on the OM play key roles in regulating the mechanical properties of the OM. However, whether the OM composition and mechanical properties are coordinated with the expression of the T3SS genes is unknown. Here, we found that the two-component system (TCS) regulator P-RhpR, a direct repressor of the T3SS regulator hrpRS operon, directly activates the expression of the OM lipoprotein gene grlp bearing a function in regulating the periplasmic space. This finding suggests a coordination between the OM properties and the T3SS gene regulation and reveals a new target for control of the T3SS gene expression and bacterial pathogenicity.

MmpA, a Conserved Membrane Protein Required for Efficient Surface Transport of Trehalose Lipids in Corynebacterineae.

Cell walls of bacteria of the genera Mycobacterium and Corynebacterium contain high levels of (coryno)mycolic acids. These very long chain fatty acids are synthesized on the cytoplasmic leaflet of the inner membrane (IM) prior to conjugation to the disaccharide, trehalose, and transport to the periplasm. Recent studies on Corynebacterium glutamicum have shown that acetylation of trehalose monohydroxycorynomycolate (hTMCM) promotes its transport across the inner membrane. Acetylation is mediated by the membrane acetyltransferase, TmaT, and is dependent on the presence of a putative methyltransferase, MtrP. Here, we identify a third protein that is required for the acetylation and membrane transport of hTMCM. Deletion of the C. glutamicum gene NCgl2761 (Rv0226c in Mycobacterium tuberculosis) abolished synthesis of acetylated hTMCM (AcTMCM), resulting in an accumulation of hTMCM in the inner membrane and reduced synthesis of trehalose dihydroxycorynomycolate (h2TDCM), a major outer membrane glycolipid. Complementation with the NCgl2761 gene, designated here as mmpA, restored the hTMCM:h2TDCM ratio. Comprehensive lipidomic analysis of the ΔtmaT, ΔmtrP and ΔmmpA mutants revealed strikingly similar global changes in overall membrane lipid composition. Our findings suggest that the acetylation and membrane transport of hTMCM is regulated by multiple proteins: MmpA, MtrP and TmaT, and that defects in this process lead to global, potentially compensatory changes in the composition of inner and outer membranes.

Outer membrane and phospholipid composition of the target membrane affect the antimicrobial potential of first- and second-generation lipophosphonoxins

Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.

Giant Vesicles Mimicking Outer Membrane of Gram-Negative Bacterium

Engineering of synthetic cells which mimics the native membrane opens up various platforms for research in membrane biology as well as synthetic biology field. In this work, giant unilamellar vesicles (GUV) mimicking outer membrane of Gram-negative bacterium is engineered by incorporating lipopolysaccharide (LPS) under physiological salt condition. We successfully incorporated LPS in Gram-negative (GN) vesicles by inverse-phase precursor film assembly using gel-assisted technology and confirmed at single-vesicle level using fluorescence detection system. Effective integration of LPS in vesicles is attained in presence of Ca2+ ions at a ratio of LPS: E. coli polar lipid extract (GN extract) (1:6) to construct outer membrane mimicking (OMM) vesicles comparable to the bacterial outer-membrane composition. Quantitative transport rates of antibiotic showed a much slower flux across vesicles with LPS (OMM) compared to GN vesicles further confirming the incorporation of LPS in vesicles and its significance in native bacterial cells. To demonstrate the functionality of engineered OMM vesicles, a model peptide melittin is reconstituted in the membrane of both vesicles and the uptake of small different sized molecules is studied. In GN vesicles, a steady uptake of large molecules is observed in peptide: lipid (1:15) ratio, where in OMM vesicles pore formation and diffusion of molecules occurred at higher peptide: lipid (1:5) ratio confirming intrinsic resistance of OMM vesicles due to presence of LPS. The OMM vesicles constructed here will find immense applications in fundamental understanding of membrane biophysical phenomena.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA)

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

Outer membrane compositions enhance the rate of extracellular electron transport via cell-surface MtrC protein in Shewanella oneidensis MR-1

While cell membrane composition is critical for the function of membrane proteins, membrane modification has not been used to control the rate of extracellular electron transfer (EET) via the outer membrane protein complexes. Here, the rate of electron flow via the cell-surface redox protein, MtrC, was highly enhanced upon change in the outer membrane composition in Shewanella oneidensis MR-1. The MR-1 strain was pre-cultured at 4 °C and 30 °C to initiate differentiation of membrane composition. The whole-cell difference electrochemical assay of wild-type and mutant strains lacking MtrC suggested that the rate of EET via MtrC increased approximately 18 times at 4 °C than 30 °C. Circular dichroism spectroscopy showed that the molar exciton coupling coefficient for inter-heme interaction in MtrC increased in MR-1 at 4 °C than 30 °C. Results suggest that membrane modification may be a novel strategy for improving the efficiency of EET-based technologies.

Determining the Role of Francisella lipoprotein A (FlpA) in Francisella tularensis Susceptibility to Resazomycins.

Antibiotic resistance is one of the top threats to global public health. In the United States, over two million people each year are infected with antibiotic resistant bacteria which results in approximately 23,000 deaths. The development of new antibiotics is essential to combat this crisis and prevent the loss of additional lives. Our laboratory discovered that resazurin exhibits antimicrobial activity against a select family of Gram-negative bacteria including Neisseria gonorrhoeae, Helicobacter pylori, and Francisella tularensis. Resazurin and derivatives of this compound, resazomycins, can kill N. gonorrhoeae and F. tularensis in broth culture and inside host cells. One resazomycin, resorufin pentyl ether, reduces vaginal colonization by N. gonorrhoeae in a mouse model of infection. To identify potential targets of resazomycins, we performed a high throughput screen to select for resazurin-resistant F. tularensis isolates and performed whole genome sequencing on each isolate. Multiple F. tularensis isolates had a mutation in the gene FTL_0073 which encodes for Francisella lipoprotein A (FlpA). We hypothesized that this protein could be a target of resazomycins or may play a role in uptake of these antibiotics. To address these hypotheses, we generated a flpA disruption mutant. First, we evaluated the susceptibility of the flpA mutant to resazomycins and preliminary data indicates this mutant is resistant. Next, we will test the flpA mutant for changes in outer membrane composition and defects in uptake of resazomycins. Understanding the role of flpA in resazomycin susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

Uncovering Biophysical Properties and Interactions of Bacteria Membrane using An Outer Membrane Supported Bilayer

Antibiotic resistance is a growing global health concern that has been increasing in prevalence in the past few decades. In Gram-negative bacteria, changes in membrane properties such as surface charge, length of membrane components such as lipopolysaccharide (LPS), and in physical properties of the membrane such as domain clustering, can alter responses to antibiotics and mediate resistance. To study the effect of these properties, model membranes have been used to mimic bacterial membranes and to study changes in membrane properties using quantitative surface techniques. However, most model membrane platforms do not capture the complexity of bacterial membranes, which also contain proteins, LPS, and exhibit asymmetry and diverse lipid compositions. We developed a substrate-supported membrane platform using outer membrane vesicles (OMVs) to characterize biophysical membrane properties and investigate the interactions of bacterial membrane and antibacterial compounds. OMVs are naturally shed from the OM by many Gram-negative bacteria from the outer membrane (OM) and recapitulate OM composition and physical properties. Using five clinical bacterial isolates, we demonstrated the formation of fluid bilayers using a membrane fluorescent dye, octadecyl rhodamine B, and measured diffusion coefficients using fluorescence recovery after photobleaching (FRAP). Using total internal reflection fluorescence microscopy (TIRFM), we also confirmed the presence of outer membrane material such as lipid A of LPS and the proper orientation and asymmetry of the bilayer using genetic modifications involving cleavable fluorescent proteins. To study interactions between bacterial membrane and antibacterial compounds, we use optical techniques to observe changes in membrane properties and quantify membrane interactions using polymyxin B. Findings from this work demonstrate OMV bilayers as a platform for studying membrane interactions while retaining membrane components and asymmetry found in native bacterial membranes.

Structural and electrochemical characterization of lawsone-dependent production of tellurium-metal nanoprecipitates by photosynthetic cells of Rhodobacter capsulatus

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus exploit the simultaneous presence in the cultural medium of the toxic oxyanion tellurite (TeO32−) and the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) by reducing tellurite to metal Te0 nanoprecipitates (TeNPs) outside the cells. Here we have studied the mechanism by which lawsone interacts with metabolically active cells and analysed both structure and composition of the TeNPs collected from the growth medium of phototrophycally grown R. capsulatus. High Resolution Transmission Electron Microscopy (HR-TEM) images and Energy-Dispersive X-ray (EDX) microanalysis of TeNPs showed a central core of polycrystalline tellurium interspersed in an organic matrix with a predominant protein-based composition. The main proteins from Te0 nanostructures were identified by Liquid Chromatography tandem-Mass Spectrometry and were all correlated with the cell outer membrane composition. The interaction of reduced lawsone with tellurite and with the bacterial cells was probed by Cyclic Voltammetry and Scanning ElectroChemical Microscopy (SECM). We concluded that lawsone is required for the reduction of tellurite to metal Te0 in a reaction mechanism dependent on reducing equivalents deriving from the cell photosynthetic metabolism. SECM experiments demonstrate that lawsone, by diffusing inside the bacterial cells, is effectively available at the membrane site of the photosynthetic electron transport chain.

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.

GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the ∆2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and a model for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.-Seib, K. L., Haag, A. F., Oriente, F., Fantappiè, L., Borghi, S., Semchenko, E. A., Schulz, B. L., Ferlicca, F., Taddei, A. R., Giuliani, M. M., Pizza, M., Delany, I. The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.

Mutagenesis of the rpoS gene involved in alteration of outer membrane composition

rpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the rpoS gene. The aim of this study was to investigate if rpoS mutagenesis has impact on bacterial morphology in addition to cell division. A 0.603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy. After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that mutation in rpoS gene leads to decomposition of outer membrane of F. chinesis. A suitable vector to make suicide mutation in rpoS was constructed for F. chinesi.

Probing Membrane Association of \u03b1-Synuclein Domains with VDAC Nanopore Reveals Unexpected Binding Pattern

It is well established that α-synuclein (α-syn) binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. Previously, we have found that a naturally occurring nanopore of the mitochondrial voltage-dependent anion channel (VDAC), reconstituted into planar bilayers of a plant-derived lipid, responds to α-syn at nanomolar solution concentrations. Here, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we show that functionally important binding does indeed take place in the nanomolar range. We demonstrate that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures α-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrates a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of α-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of α-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of α-syn capture by the nanopore is not simply proportional to the α-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore.

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as “core” proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. SignificanceYersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics.

Deprivation of the Periplasmic Chaperone SurA Reduces Virulence and Restores Antibiotic Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the β-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Swarmer Cell Development of the Bacterium Proteus mirabilis Requires the Conserved Enterobacterial Common Antigen Biosynthesis Gene rffG.

Individual cells of the bacterium Proteus mirabilis can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating that P. mirabilis requires the gene rffG for swarmer cell elongation and subsequent swarm motility. The rffG gene encodes a protein homologous to the dTDP-glucose 4,6-dehydratase protein of Escherichia coli, which contributes to enterobacterial common antigen biosynthesis. Here, we characterize the rffG gene in P. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that the absence of the rffG gene induces several stress response pathways, including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that in rffG-deficient cells, the suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, the loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that an RcsB-mediated response acts to suppress the emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.IMPORTANCEProteus mirabilis swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multifunctional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the rffG-dependent moieties provide a novel attractive target for potential antimicrobials.