The Gram-positive cell wall consists of peptidoglycan functionalized with anionic glycopolymers, such as wall teichoic acid and capsular polysaccharide (CP). How the different cell wall polymers are ...assembled in a coordinated fashion is not fully understood. Here, we reconstitute Staphylococcus aureus CP biosynthesis and elucidate its interplay with the cell wall biosynthetic machinery. We show that the CapAB tyrosine kinase complex controls multiple enzymatic checkpoints through reversible phosphorylation to modulate the consumption of essential precursors that are also used in peptidoglycan biosynthesis. In addition, the CapA1 activator protein interacts with and cleaves lipid-linked CP precursors, releasing the essential lipid carrier undecaprenyl-phosphate. We further provide biochemical evidence that the subsequent attachment of CP is achieved by LcpC, a member of the LytR-CpsA-Psr protein family, using the peptidoglycan precursor native lipid II as acceptor substrate. The Ser/Thr kinase PknB, which can sense cellular lipid II levels, negatively controls CP synthesis. Our work sheds light on the integration of CP biosynthesis into the multi-component Gram-positive cell wall.
Summary
Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. ...The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross‐linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well‐studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan.
Summary
Peptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria ...replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.
Obligate intracellular bacteria are under selective pressure to reduce the abundance or detection of their highly immunostimulatory cell wall constituent peptidoglycan. In this work we have performed a comparative bioinformatics analysis to identify genes involved in peptidoglycan metabolism that have been retained or lost in different obligate intracellular bacteria. We identify a group of organisms unified in the presence/absence of a subset of key genes, and predict them as likely to have minimal peptidoglycan‐like structures.
Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections. ...Here, by applying native mass spectrometry, we elucidate the effects of lipid substrates on the PG membrane enzymes MraY, MurG, and MurJ. We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C
-P). Further, we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes. Overall, we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention.
The Firmicutes are a phylum of bacteria that dominate numerous polymicrobial habitats of importance to human health and industry. Although these communities are often densely colonized, a broadly ...distributed contact-dependent mechanism of interbacterial antagonism utilized by Firmicutes has not been elucidated. Here we show that proteins belonging to the LXG polymorphic toxin family present in
mediate cell contact- and Esx secretion pathway-dependent growth inhibition of diverse Firmicute species. The structure of one such toxin revealed a previously unobserved protein fold that we demonstrate directs the degradation of a uniquely bacterial molecule required for cell wall biosynthesis, lipid II. Consistent with our functional data linking LXG toxins to interbacterial interactions in
, we show that LXG genes are prevalent in the human gut microbiome, a polymicrobial community dominated by Firmicutes. We speculate that interbacterial antagonism mediated by LXG toxins plays a critical role in shaping Firmicute-rich bacterial communities.
Activities and regulation of peptidoglycan synthases Egan, Alexander J. F.; Biboy, Jacob; van't Veer, Inge ...
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
10/2015, Volume:
370, Issue:
1679
Journal Article
Peer reviewed
Open access
Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the ...cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.
Regulation of bacterial cell wall growth Egan, Alexander J. F.; Cleverley, Robert M.; Peters, Katharina ...
The FEBS journal,
March 2017, Volume:
284, Issue:
6
Journal Article
Peer reviewed
Open access
During growth and propagation, a bacterial cell enlarges and subsequently divides its peptidoglycan (PG) sacculus, a continuous mesh‐like layer that encases the cell membrane to confer mechanical ...strength and morphological robustness. The mechanism of sacculus growth, how it is regulated and how it is coordinated with other cellular processes is poorly understood. In this article, we will discuss briefly the current knowledge of how cell wall synthesis is regulated, on multiple levels, from both sides of the cytoplasmic membrane. According to the current knowledge, cytosolic scaffolding proteins connect PG synthases with cytoskeletal elements, and protein phosphorylation regulates cell wall growth in Gram‐positive species. PG‐active enzymes engage in multiple protein–protein interactions within PG synthesis multienzyme complexes, and some of the interactions modulate activities. PG synthesis is also regulated by central metabolism, and by PG maturation through the action of PG hydrolytic enzymes. Only now are we beginning to appreciate how these multiple levels of regulating PG synthesis enable the cell to propagate robustly with a defined cell shape under different and variable growth conditions.
We review how bacterial cell wall growth is regulated on multiple levels. We outline the assembly of synthesis complexes through protein scaffolding and how dynamic protein–protein interactions control cell wall growth. We also discuss other regulatory inputs including phosphorylation of key regulatory proteins, cell wall maturation and coordination of cell wall growth with central metabolism through sensing nutrient availability.
Mechanical interactions between bacterial cells and extracellular polymeric substance are essential in determining biofilm assembly and disassembly as well the mechanical characteristics of biofilms. ...However, the physics of these mechanical interactions in different cell culture conditions are poorly understood. We created typical artificial biofilm consisting of planktonic bacteria and hydrogel, in the absence of metabolic or regulatory effect. We have demonstrated that the cell culture medium can significantly affect the mechanical interactions between bacterial cells and hydrogels. The stiffness of the bacteria-hydrogel artificial biofilm cannot be simply attributed by the summation of the contribution from the bacteria and hydrogel based on the mathematical models and computational models. We have revealed that the tryptone component of Luria-Bertani broth medium plays an important role in stiffening effect of bacteria-hydrogel construct. Such significant stiffening effect can be explained by the following mechanism: the presence of tryptone in cell culture medium may enable the bacteria itself to crosslink the hydrogel polymer chains. Our findings have also demonstrated the synergy of modelling and innovative experiments which would potentially impact the biofilm control strategies.
During the transition from elongation to septation, Escherichia coli establishes a ring-like peptidoglycan growth zone at the future division site. This preseptal peptidoglycan synthesis does not ...require the cell division-specific peptidoglycan transpeptidase PBP3 or most of the other cell division proteins, but it does require FtsZ, its membrane-anchor ZipA and at least one of the bi-functional transglycosylase-transpeptidases, PBP1A or PBP1B. Here we show that PBP1A and PBP1B interact with ZipA and localise to preseptal sites in cells with inhibited PBP3. ZipA stimulates the glycosyltransferase activity of PBP1A. The membrane-anchored cell division protein FtsN localises at preseptal sites and stimulates both activities of PBP1B. Genes zipA and ftsN can be individually deleted in ftsA* mutant cells, but the simultaneous depletion of both proteins is lethal and cells do not establish preseptal sites. Our data support a model according to which ZipA and FtsN-FtsA have semi-redundant roles in connecting the cytosolic FtsZ ring with the membrane-anchored peptidoglycan synthases during the preseptal phase of envelope growth.
Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several ...essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the "on state" of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.
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