Transposon Screen of Surface Accessibility in S. aureus

Bacterial cell walls represent one of the most prominent targets of antibacterial agents. These agents include natural products (e.g., vancomycin) and proteins stemming from the innate immune system (e.g., peptidoglycan-recognition proteins and lysostaphin). Among bacterial pathogens that infect humans, Staphylococcus aureus (S. aureus) continues to impose a tremendous healthcare burden across the globe. S. aureus has evolved countermeasures that can directly restrict the accessibility of innate immune proteins, effectively protecting itself from threats that target key cell well components. We recently described a novel assay that directly reports on the accessibility of molecules to the peptidoglycan layer within the bacterial cell wall of S. aureus. The assay relies on site-specific chemical remodeling of the peptidoglycan with a biorthogonal handle. Here, we disclose the application of our assay to a screen of a nonredundant transposon mutant library for susceptibility of the peptidoglycan layer with the goal of identifying genes that contribute to the control of cell surface accessibility. We discovered several genes that resulted in higher accessibility levels to the peptidoglycan layer and showed that these genes modulate sensitivity to lysostaphin. These results indicate that this assay platform can be leveraged to gain further insight into the biology of bacterial cell surfaces.Table of Contents Figure

Prospects for the Mechanism of Spiroplasma Swimming

Spiroplasma are helical bacteria that lack a peptidoglycan layer. They are widespread globally as parasites of arthropods and plants. Their infectious processes and survival are most likely supported by their unique swimming system, which is unrelated to well-known bacterial motility systems such as flagella and pili. Spiroplasma swims by switching the left- and right-handed helical cell body alternately from the cell front. The kinks generated by the helicity shift travel down along the cell axis and rotate the cell body posterior to the kink position like a screw, pushing the water backward and propelling the cell body forward. An internal structure called the “ribbon” has been focused to elucidate the mechanisms for the cell helicity formation and swimming. The ribbon is composed of Spiroplasma-specific fibril protein and a bacterial actin, MreB. Here, we propose a model for helicity-switching swimming focusing on the ribbon, in which MreBs generate a force like a bimetallic strip based on ATP energy and switch the handedness of helical fibril filaments. Cooperative changes of these filaments cause helicity to shift down the cell axis. Interestingly, unlike other motility systems, the fibril protein and Spiroplasma MreBs can be traced back to their ancestors. The fibril protein has evolved from methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase, which is essential for growth, and MreBs, which function as a scaffold for peptidoglycan synthesis in walled bacteria.

Growth and Division of the Peptidoglycan Matrix

Most bacteria are surrounded by a peptidoglycan cell wall that defines their shape and protects them from osmotic lysis. The expansion and division of this structure therefore plays an integral role in bacterial growth and division. Additionally, the biogenesis of the peptidoglycan layer is the target of many of our most effective antibiotics. Thus, a better understanding of how the cell wall is built will enable the development of new therapies to combat the rise of drug-resistant bacterial infections. This review covers recent advances in defining the mechanisms involved in assembling the peptidoglycan layer with an emphasis on discoveries related to the function and regulation of the cell elongation and division machineries in the model organisms Escherichia coli and Bacillus subtilis. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

Interactions of surfactants with the bacterial cell wall and inner membrane: Revealing the link between aggregation and antimicrobial activity

Surfactants with their intrinsic ability to solubilize lipids are widely used as antibacterial agents. Interaction of surfactants with the bacterial cell envelope is complicated due to their propensity to aggregate. It is important to discern the interactions of micellar aggregates and single surfactants on the various components of the cell envelope to improve selectivity and augment the efficacy of surfactant-based products. In this study, we present a combined experimental and molecular dynamics investigation to unravel the molecular basis for the superior kill efficacy of laurate over oleate observed in contact time assays with live E. coli. To gain a molecular understanding of these differences, we performed all-atom molecular dynamics simulations to observe the interactions of surfactants with the periplasmic peptidoglycan layer and the inner membrane of Gram-negative bacteria. The peptidoglycan layer allows a greater number of translocation events for laurate when compared with oleate molecules. More interestingly, aggregates did not translocate the peptidoglycan layer, thereby revealing an intrinsic sieving property of the bacterial cell wall to effectively modulate the surfactant concentration at the inner membrane. The molecular dynamics simulations exhibit greater thinning of the inner membrane in the presence of laurate when compared with oleate, and laurate induced greater disorder and decreased the bending modulus of the inner membrane to a greater extent. The enhanced antimicrobial efficacy of laurate over oleate was further verified by experiments with giant unilamellar vesicles, which revealed that laurate induced vesicle rupture at lower concentrations in contrast to oleate. The novel molecular insights gained from our study uncovers hitherto unexplored pathways to rationalize the development of antimicrobial formulations and therapeutics.

Exiguobacterium Sp. HA2, isolated from the Ilam Mountains of Iran

ABSTRACTA motile, Gram-stain-positive, rod-shaped, non-sporing, tolerate up to 5% NaCl, grew at 0–25 °C, designated Exiguobacterium sp. HA2 was isolated from the soil of the Ilam Mountains of Iran during October 2016. The major isoprenoid quinone is MK-7 and in the smaller amount are MK-6 and MK-8. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine. Major fatty acids (>10 %) are isoC13:0, isoC15:0 and C16:0. The bacterial cell wall peptidoglycan layer was lysine-glycine. The 16S rRNA sequence was analyzed at the phylogenetic levels. Also, A supplemental comparison was made between five other genes including csp, gyrB, hsp70, rpoB, and citC. According to the results of genotypic and phenotypic characteristics, the strain was categorized in the genus Exiguobacterium. This bacterium had the closest relation with Exiguobacterium undae, and thus was dubbed Exiguobacterium sp. HA2. The different in the Phenotypic, functional characteristics and phylogenetic indicated Exiguobacterium sp. HA2 can be regarded as representing considered a novel species within the genus Exiguobacterium.

The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Anchors the Peptidoglycan Layer by Spanning It with All Subunits

Gram-negative bacteria possess a three-layered envelope composed of an inner membrane, surrounded by a peptidoglycan (PG) layer, enclosed by an outer membrane. The envelope ensures protection against diverse hostile milieus and offers an effective barrier against antibiotics. The layers are connected to each other through many protein interactions. Bacteria evolved sophisticated machineries that maintain the integrity and the functionality of each layer. The β-barrel assembly machinery (BAM), for example, is responsible for the insertion of the outer membrane integral proteins including the lipopolysaccharide transport machinery protein LptD. Labelling bacterial cells with BAM-specific fluorescent antibodies revealed the spatial arrangement between the machinery and the PG layer. The antibody detection of each BAM subunit required the enzymatic digestion of the PG layer. Enhancing the spacing between the outer membrane and PG does not abolish this prerequisite. This suggests that BAM locally sets the distance between OM and the PG layer. Our results shed new light on the local organization of the envelope.

Analyses of the Effect of Peptidoglycan on Photocatalytic Bactericidal Activity Using Different Growth Phases Cells of Gram-Positive Bacterium and Spheroplast Cells of Gram-Negative Bacterium

We conducted photocatalytic experiments focusing on the peptidoglycan layer to elucidate the details of the mechanism of photocatalytic sterilization. The previous study of our laboratory suggested that the presence of the peptidoglycan layer increases the bactericidal effect. To further verify it, the following experiments were performed: experiments on cells with different peptidoglycan layer thickness used Lactobacillus plantarum cells with different growth phases, experiments on cells with the thin peptidoglycan layer used Escherichia coli cells and spheroplast cells from which the peptidoglycan layer was removed from E. coli cells. The bactericidal effects increased as the growth progresses of L. plantarum. It was confirmed by TEM that the thickness of the peptidoglycan layer increased with cell growth. The survival rates of E. coli intact cells were significantly lower than those of spheroplast cells. These results strongly suggest that the peptidoglycan layer enhances the photocatalytic bactericidal effect. As a result of allowing the photocatalytic reaction to act on peptidoglycan, the amount of hydroxyl radical was smaller, and the amount of hydrogen peroxide was higher than in the absence of peptidoglycan. It is suggested that peptidoglycan may convert produced hydroxyl radical to hydrogen peroxide.

The periplasmic space cannot be artificially enlarged due to homeostatic regulation maintaining spatial constraints essential for membrane spanning processes and cell viability

ABSTRACTThe cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope dictate protein and lipid transport and drug resistance phenotypes, and depend on spatial constraints across the envelope and load-bearing forces across the cell surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change periplasmic width and spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. E. coli expressing elongated Lpp activate potent homeostatic mechanisms to enforce a wild-type spatial constraint: they increase steady-state levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and depend on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Our findings demonstrate complex regulatory mechanisms for tight control over periplasmic width to enable spatial constraint essential for membrane spanning processes. They further show that the periplasm cannot be widened by engineering approaches, with implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation.