Tuning spherical cells into kinking helices in wall-less bacteria

In bacteria, cell shape is determined and maintained through a complex interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Spiroplasma species, members of the Mollicutes class, challenge this general understanding because they are characterized by a helical cell shape and motility without a cell wall. This specificity is thought to rely on five MreB isoforms and a specific fibril protein. In this study, combinations of these five MreBs and of the fibril from Spiroplasma citri were expressed in another Mollicutes, Mycoplasma capricolum. Mycoplasma cells that were initially pleomorphic, mostly spherical, turned into helices when MreBs and fibrils were expressed in this heterologous host. The fibril protein was essential neither for helicity nor for cell movements. The isoform MreB5 had a special role as it was sufficient to confer helicity and motility to the mycoplasma cells. Cryo-electron microscopy confirmed the association of MreBs and fibril-based cytoskeleton with the plasma membrane, suggesting a direct effect on the membrane curvature. Finally, the heterologous expression of these proteins, MreBs and fibril, made it possible to reproduce the kink-like motility of spiroplasmas without providing the ability of cell movement in liquid broth. We suggest that other Spiroplasma components, not yet identified, are required for swimming, a hypothesis that could be evaluated in future studies using the same model.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal and human bacterial pathogens. This multi-protein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed as pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in Dickeya, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic Homology Region domain that interacts directly with the N-terminal secretin domain. Site-specific photo cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB scaffolds the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or a pilotin GspS.

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.

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

The peptidoglycan cell wall is a predominant defining structure of bacteria, determining cell shape and supporting survival in diverse conditions. As a single, macromolecular sacculus enveloping the bacterial cell during growth and division, peptidoglycan is necessarily a dynamic structure that requires highly regulated synthesis of new material, remodeling, and turnover, or autolysis, of old material. Despite ubiquitous clinical exploitation of peptidoglycan synthesis as an antibiotic target, much remains unknown about how bacteria modulate synthetic and autolytic processes. Here, we couple bacterial genetics in <em>Vibrio cholerae</em> with compositional analysis of soluble pools of peptidoglycan turnover products to uncover a critical role for a widely misunderstood class of autolytic enzymes, the lytic transglycosylases (LTGs). We demonstrate that LTG activity is specifically required for vegetative growth. The vast majority of LTGs, however, are dispensable for growth, and defects that are ultimately lethal accumulate due to generally inadequate LTG activity, rather than the absence of specific individual enzymes. Consistent with this, we found that a heterologously expressed <em>E. coli</em> LTG, MltE, is capable of sustaining <em>V. cholerae</em> growth in the absence of endogenous LTGs. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains accumulate in the WT, and, to a higher degree, in LTG mutants, and that LTG mutants are hyper-susceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that a key function of LTGs is to prevent toxic crowding of the periplasm with synthesis-derived PG fragments. Contrary to prevailing models, our data further suggest that this process can be temporally separate from peptidoglycan synthesis.

How Teichoic Acids Could Support a Periplasm in Gram-Positive Bacteria, and Let Cell Division Cheat Turgor Pressure

The cytoplasm of bacteria is maintained at a higher osmolality than the growth medium, which generates a turgor pressure. The cell membrane (CM) cannot support a large turgor, so there are two possibilities for transferring the pressure to the peptidoglycan cell wall (PGW): (1) the CM could be pressed directly against the PGW, or (2) the CM could be separated from the PGW by a periplasmic space that is isoosmotic with the cytoplasm. There is strong evidence for gram-negative bacteria that a periplasm exists and is isoosmotic with the cytoplasm. No comparable studies have been done for gram-positive bacteria. Here I suggest that a periplasmic space is probably essential in order for the periplasmic proteins to function, including especially the PBPs that remodel the peptidoglycan wall. I then present a semi-quantitative analysis of how teichoic acids could support a periplasm that is isoosmotic with the cytoplasm. The fixed anionic charge density of teichoic acids in the periplasm is ∼0.5 M, which would bring in ∼0.5 M Na+ neutralizing ions. This approximately balances the excess osmolality of the cytoplasm that would produce a turgor pressure of 19 atm. The 0.5 M fixed charge density is similar to that of proteoglycans in articular cartilage, suggesting a comparability ability to support pressure. An isoosmotic periplasm would be especially important for cell division, since it would allow CM constriction and PGW synthesis to avoid turgor pressure.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

ABSTRACTThe peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltG’s ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

Contact-dependent traits in Pseudomonas syringae B728a

Production of the biosurfactant syringafactin by the plant pathogen Pseudomonas syringae B728a is a surface contact-dependent trait. Expression of syfA, as measured using a gfp reporter gene fusion was low in planktonic cells in liquid cultures but over 4-fold higher in cells immobilized on surfaces as varied as glass, plastic, paper, parafilm, agar, membrane filters, and leaves. Induction of syfA as measured by GFP fluorescence was rapid, occurring within two hours after immobilization of cells on surfaces. Comparison of the global transcriptome by RNA sequencing of planktonic cells in a nutrient medium with that of cells immobilized for 2 hours on filters placed on this solidified medium revealed that, in addition to syfA, 3156 other genes were differentially expressed. Genes repressed in immobilized cells included those involved in quaternary ammonium compound (QAC) metabolism and transport, compatible solute production, carbohydrate metabolism and transport, organic acid metabolism and transport, phytotoxin synthesis and transport, amino acid metabolism and transport, and secondary metabolism. Genes induced in immobilized cells included syfA plus those involved in translation, siderophore synthesis and transport, nucleotide metabolism and transport, flagellar synthesis and motility, lipopolysaccharide (LPS) synthesis and transport, energy generation, transcription, chemosensing and chemotaxis, replication and DNA repair, iron-sulfur proteins, peptidoglycan/cell wall polymers, terpenoid backbone synthesis, iron metabolism and transport, and cell division. That many genes are rapidly differentially expressed upon transfer of cells from a planktonic to an immobilized state suggests that cells experience the two environments differently. It seems possible that surface contact initiates anticipatory changes in P. syringae gene expression, which enables rapid and appropriate physiological responses to the different environmental conditions such as might occur in a biofilm. Such responses could help cells survive transitions from aquatic habitats fostering planktonic traits to attachment on surfaces, conditions that alternatively occur on leaves.

Bacterial Cell Wall Analogue Peptides Control the Oligomeric States and Activity of the Glycopeptide Antibiotic Eremomycin: Solution NMR and Antimicrobial Studies

For some time, glycopeptide antibiotics have been considered the last line of defense against Methicillin-resistant Staphylococcus aureus (MRSA). However, vancomycin resistance of Gram-positive bacteria is an increasingly emerging worldwide health problem. The mode of action of glycopeptide antibiotics is essentially the binding of peptidoglycan cell-wall fragments terminating in the d-Ala-d-Ala sequence to the carboxylate anion binding pocket of the antibiotic. Dimerization of these antibiotics in aqueous solution was shown to persist and even to enhance the antibacterial effect in a co-operative manner. Some works based on solid state (ss) Nuclear Magnetic Resonance (NMR) studies questioned the presence of dimers under the conditions of ssNMR while in a few cases, higher-order oligomers associated with contiguous back-to-back and face-to-face dimers were observed in the crystal phase. However, it is not proved if such oligomers persist in aqueous solutions. With the aid of 15N-labelled eremomycin using 15N relaxation and diffusion NMR methods, we observed tetramers and octamers when the N-Ac-d-Ala-d-Ala dipeptide was added. To the contrary, the N-Ac-d-Ala or (N-Ac)2-l-Lys-d-Ala-d-Ala tripeptide did not induce higher-order oligomers. These observations are interesting examples of tailored supramolecular self-organization. New antimicrobial tests have also been carried out with these self-assemblies against MRSA and VRE (resistant) strains.