In order to facilitate PG recycling, the product MurNAc-6P-GlcNAc is split intracellularly by a novel phospho-glycosidase MupG , constituting the first characterized representative of a novel class of phospho-muramidase enzymes distributed mainly within the Firmicutes bacteria.
Hager et al. In this study, the enzymatic pathway leading to the synthesis of pyruvylated disaccharide repeats, [beta-GlcNAc-1,3- 4,6-Pyr -beta-ManNAc], of the P. The reconstitution involved recombinant CsaB enzyme, catalyzing the attachment of a pyruvate to position 4 and 6 of ManNAc in the lipid-linked precursor molecule.
Devine provides a concise mini-review about the phosphate starvation regulation in the Gram-negative E. In both organisms, phosphate limitation is sensed by the two-component system PhoPR. However, the mechanisms controlling the Pho response differ. In Bacillus subtilis , phosphate-limitation response is linked with wall teichoic acid metabolism.
PhoR activity is controlled by biosynthetic intermediates of WTA metabolism, which either promotes or inhibits autokinase activity.
Vermassen et al. The unique chemical nature of PG allows it to act as a potent signaling molecule. Irazoki et al. The authors highlight the multiplicity of systems to generate and sense bacterial PG and suggest that there is still a great deal to be learned about the sensing of these important molecules.
They conclude that this field will be driven by the development and application of new analytical technologies to identify novel PG receptors. Peptidoglycan recycling among many Gram-negative bacteria is achieved through a core pathway of degradation, recovery and recycling.
In some pathogenic Neisseria , the recycling system is partially defective, which leads to an increase in the release of immunostimulatory PG fragments. In their review article, Schaub and Dillard discuss some of the differences between Neisserial PG turnover and other, more intensively studied bacteria such as E.
They conclude by proposing Neisseria sp. Sychanta et al. They also discuss current efforts at understanding the impact of inhibiting these systems and address unanswered biological questions such as the source of acetate for wall modification. Bacterial cell wall biology remains a major frontier, both in our quest to develop a profound understanding of fundamental microbiology and to discover novel compounds that may be used to treat infections caused by antibiotic resistant bacteria.
We hope that this special issue further advances this frontier and inspires additional exploration—peptidoglycan is, in many ways, still as mysterious as it was 7, publications ago. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Cava, F. Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. Cho, H. P, Rohs, D. Fleming, T. Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease.
PubMed Abstract Google Scholar. Goldman, W. Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells. Jankute, M. Assembly of the mycobacterial cell wall. Johnson, J. Bacterial cell-wall recycling.
Jutras, B. Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis. In Gram-positive bacteria, most of these proteins are secreted by the universally conserved and essential Sec pathway. This pathway has been extensively studied in E.
Almost all proteins that are targeted by this secretory pathway have an N-terminal signal peptide composed of approximately 30 amino acids. Once the proteins have been translocated across the cytoplasmic membrane, this signal peptide is cleaved off by the appropriate signal peptidase.
Then, the protein is either released into the extracellular medium or, alternatively, it is retained in the cell envelope, if it contains a specific sequence ensuring its attachment to the cytoplasmic membrane or the components of the cell wall in addition to the signal peptide.
Secreted proteins can be covalently attached to the cell surface by sortase-mediated reactions or non-covalently attached via i transmembrane anchors; ii lipid anchors; or iii different cell wall binding domains CWBD [ , ]. We will review here LAB proteins which are linked to cell wall components through covalent or non covalent binding.
A portion of a given cell wall protein is covalently bound to PG by a transpeptidation mechanism that is catalyzed by sortase A SrtA, also called housekeeping sortase. In addition to an N-terminal signal peptide, they also contain, at their C-terminal, a conserved LPXTG motif that is followed by a stretch of hydrophobic residues and a positively charged tail [ , , ].
Inactivation of srtA in L. These proteins contain mucus-binding domains MUB or MucBP that are thought to play an important role in the adhesion of LAB to the mucus layer that covers intestinal epithelial cells [ ].
Other functionally important LPXTG proteins are the pilins, which are the structural components of pili. Pili or fimbriae are long filamentous structures that extend from the surfaces of various Gram-negative and Gram-positive bacteria.
Most studies on pili in Gram-positive bacteria have been conducted on pathogenic species, including streptococci, enterococci, corynebacteria, and bacilli [ — ]. Pili have been shown to be involved in adhesion to host cells and tissues and are thus considered to promote host colonization and invasion [ ]. In Gram-positive bacteria, the sortase-dependent pili Spa-type for sortase-mediated pilus assembly are composed of a major backbone pilin, whose subunits are covalently assembled by sortase C, and of one or two accessory pilins.
The minor pilins are located at the base and the tip of the pilus and are possibly also dispersed along the shaft. The pili structures are anchored to PG by housekeeping sortase A [ ]. The presence of pili in LAB and in bifidobacteria has also been described and has been linked to the ability of these bacteria to colonize the guts of their hosts and persist in their gastrointestinal tracts [ , ]. More recently, L. In a natural L. This strain produces thin pili that are rather short averaging nm length.
These proteins contain specific CWBDs that have been described in several reviews [ , ]. The LysM sequence Lys motif, PF is about 40 amino acid residues long and is present in more than 2, eukaryotic and prokaryotic proteins. Several LysM sequences linked by intervening sequences constitute a LysM domain [ 70 , , ].
Studies examining the binding patterns of different PG chemotypes have found that LysM non-covalently binds to the GlcNAc moiety of glycan chains [ 70 ]. The presence of an optimal number of LysM sequences is crucial for the enzymatic activity of PGHs and, as a consequence, for the different functions of these bacterial enzymes: they are involved in cell growth, cell separation, and autolysis.
The main lactococcal autolysin AcmA, which is one of the best studied PGHs, has a modular structure and a C-terminal LysM domain that contains three LysM sequences and an N-terminal N-acetyl-glucosaminidase catalytic domain [ 68 ]. AcmA also binds to PG in other bacteria, and even to the cells of different Gram-positive species in mixed communities [ ]. It has been shown that, in L. In the case of the lactococcal autolysin AcmA, it has been proposed that attachment to the cell wall can be hindered by CW constituents; LTAs are suggested candidates [ 70 , ].
This domain is the bacterial equivalent of the well-characterized SH3 domain that is found in eukaryotes and viruses. Conflicting results have been obtained when it comes to the PG motif recognized by this domain. In staphylococci with a five-Gly PG crossbridge, the length and amino acid composition of the cross-bridge have been found to have a significant impact on the binding of the SH3-containing homolog of lysostaphin ALE-1 [ ].
However, more recently, single-molecule AFM experiments using tips functionalized with the L. This domain was initially identified based on in silico analysis of gene clusters that encode the cell surface proteins of lactobacilli, enterococcoci, and listeria species [ ]. Proteins containing the WxL domain are present in L. This domain was recently discovered in the C-terminal of the endolysins Lc-Lys and Lc-Lys2 of prophages found in the complete genome sequence of L.
It does not share amino acid sequence similarity with any known CWBDs. The domain can bind to PG and can specifically recognize the amidated D-Asp cross-bridge that occurs in L. This domain is also present in the endolysins of other L.
The surface S layer entirely coats the bacterial surface and is composed of glyco proteins that intrinsically form a two-dimensional paracrystalline structure. Most prokaryotic S-layer proteins possess a signal peptide.
These proteins bind non-covalently via their N- or C-terminus to PG or secondary cell wall polymers. The attachment is mediated by S-layer homologous domains SLHDs , which can also be found in other enzymes of Gram-positive bacteria [ , ]. S-layer proteins are present in lactobacilli, and their structure and functions have already been extensively reviewed [ ]. The cell wall ligands of the S-layer proteins isolated from different Lactobacillus species have been proposed to be carbohydrates either teichoic acids or neutral polysaccharides [ ].
The surface proteins of probiotic or commensal bacteria are thought to facilitate mucosal colonization and persistence in the gastrointestinal tract; they may also favor cross-talk with immune cells by mediating direct contact with the intestinal mucosa.
The role of pili appendages and mucus-binding proteins as surface determinants in certain LAB strains has been underscored: they allow bacteria to adhere to intestinal epithelial cells or mucus. Notably, the pili identified in L. Furthermore, pili synthetized by a natural isolate of L.
Two types of surface determinants--pili and mucus-binding proteins--have also been shown to play a role in bacterial adhesion to model mucins, and mucus-binding proteins make a greater contribution under shear flow conditions [ ]. Moreover, L. Other cell wall-associated or secreted proteins of probiotic strains have also been shown to be involved in modulating the response of the host immune system.
The attachment of bacteria to DCs has been shown to stimulate immature DCs and regulate T-cell function. Also, p40 and p75, two proteins described above section 1. From an applied perspective, LAB, because of their GRAS status, are considered to be convenient vectors for delivering therapeutic proteins or antigens to gastrointestinal tract mucosa.
In an alternative approach to vector creation that avoids the use of genetically modified bacteria, proteins of interest can be fused with CWBDs found in cell wall proteins and then anchored on the surfaces of LAB. The ability of LysM and SLH domains to bind to bacterial cell walls has been exploited to display protein antigens on LAB surfaces when developing oral vaccines [ , ].
Remarkable advances have been made in the last two decades in terms of understanding the structure and function of LAB cell walls. Cell wall components have been purified from several LAB species, which has allowed the elucidation of fine-scale cell wall structure as well as interspecific and intraspecific variation.
In tandem, genes involved in cell wall biosynthesis, modification, and degradation pathways have been identified, which has allowed for the construction of mutants that can be used to investigate the role of such genes in bacterial physiology; the results obtained can also inform technological and health applications of LAB.
Specific progress has been made with regards to deciphering the molecular mechanisms that control PGH activity and bacterial autolysis, the anchoring of cell wall proteins on the bacterial surface, the adsorption of bacteriophages to the target bacterial surface, and the cross-talk between probiotic bacteria and host cells. The results obtained have underscored the importance of further investigating LAB cell wall structure and function and thus expanding into new directions of research.
Novel structural modifications of PG have been identified, along with the genes that are involved. However, the role of these modifications in bacterial physiology, their distribution along the inner cell wall, and their influence on bacteria-host interactions remain to be investigated in detail. Furthermore, the enzymes responsible for PG modifications, such as O -acetyltransferase OatA [ 44 ], may be working in concert with other proteins that are involved in cell division, proteins that remain to be identified.
The function of such modifications needs to be investigated further in other LAB species and its role in bacteria-host interactions should be characterized. However, WPSs are also present, and they are essential for the proper septation and division of bacterial cells, which indicates that they probably play a crucial role in maintaining cell wall architecture and integrity.
Nonetheless, their exact function has not yet been deciphered. Further work should aim to identify both WPS binding sites on PG as well as the enzymes involved in creating the covalent bonds. The full range of WPS activity and the control that these molecules exert over cell wall protein localization also require further investigation. The arrangement of the different polymers inside the cell wall remains largely unknown. AFM has already proven to be a powerful technique with which to explore bacterial surface architecture at the nanoscale.
Topographic imaging of the surface of several LAB, including L. In addition, single-molecule force spectroscopy may be used to explore the spatial distribution and molecular elasticity of such structures. The structural diversity that exists in cell wall components among bacterial species and strains may underlie strain-dependent differences in processes such as autolysis and characteristics such as stress resistance, probiotic properties, or phage sensitivity; consequently, this diversity merits further study.
For instance, a better understanding of interstrain structural variation in L. From an applied perspective, a more thorough comprehension of the molecular mechanisms behind phage adsorption on host bacteria should allow us to design practical strategies to fight phage infections.
A first series of successes has stemmed from the characterization of the cell wall determinants involved in interactions between probiotic bacteria and host cells. The next step is to identify the host cell receptors that are involved in the recognition of cell wall components and the signal transduction pathways that lead to cell response.
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Studies have actually shown that the internal pressure of a cell is similar to the pressure found inside a fully inflated car tire. That is a lot of pressure for the plasma membrane to withstand! The cell wall can keep out certain molecules, such as toxins, particularly for gram negative bacteria. And lastly, the bacterial cell wall can contribute to the pathogenicity or disease —causing ability of the cell for certain bacterial pathogens.
Let us start with peptidoglycan, since it is an ingredient that both bacterial cell walls have in common. The chains are cross-linked to one another by a tetrapeptide that extends off the NAM sugar unit, allowing a lattice-like structure to form. The four amino acids that compose the tetrapeptide are: L-alanine, D-glutamine, L-lysine or meso -diaminopimelic acid DPA , and D-alanine.
Typically only the L-isomeric form of amino acids are utilized by cells but the use of the mirror image D-amino acids provides protection from proteases that might compromise the integrity of the cell wall by attacking the peptidoglycan. In many gram positive bacteria there is a cross-bridge of five amino acids such as glycine peptide interbridge that serves to connect one tetrapeptide to another. In either case the cross-linking serves to increase the strength of the overall structure, with more strength derived from complete cross-linking , where every tetrapeptide is bound in some way to a tetrapeptide on another NAG-NAM chain.
While much is still unknown about peptidoglycan, research in the past ten years suggests that peptidoglycan is synthesized as a cylinder with a coiled substructure, where each coil is cross-linked to the coil next to it, creating an even stronger structure overall.
The cell walls of gram positive bacteria are composed predominantly of peptidoglycan. The NAM tetrapeptides are typically cross-linked with a peptide interbridge and complete cross-linking is common. All of this combines together to create an incredibly strong cell wall. The additional component in a gram positive cell wall is teichoic acid , a glycopolymer, which is embedded within the peptidoglycan layers.
Teichoic acid is believed to play several important roles for the cell, such as generation of the net negative charge of the cell, which is essential for development of a proton motive force. Teichoic acid contributes to the overall rigidity of the cell wall, which is important for the maintenance of the cell shape, particularly in rod-shaped organisms.
There is also evidence that teichoic acids participate in cell division, by interacting with the peptidoglycan biosynthesis machinery. Teichoic acids can either be covalently linked to peptidoglycan wall teichoic acids or WTA or connected to the cell membrane via a lipid anchor, in which case it is referred to as lipoteichoic acid.
Since peptidoglycan is relatively porous, most substances can pass through the gram positive cell wall with little difficulty. But some nutrients are too large, requiring the cell to rely on the use of exoenzymes. The cell walls of gram negative bacteria are more complex than that of gram positive bacteria, with more ingredients overall.
What is most notable about the gram negative cell wall is the presence of a plasma membrane located outside of the peptidoglycan layers, known as the outer membrane. This makes up the bulk of the gram negative cell wall.
The outer membrane is composed of a lipid bilayer, very similar in composition to the cell membrane with polar heads, fatty acid tails, and integral proteins. It differs from the cell membrane by the presence of large molecules known as lipopolysaccharide LPS , which are anchored into the outer membrane and project from the cell into the environment.
LPS is made up of three different components: 1 the O-antigen or O-polysaccharide , which represents the outermost part of the structure , 2 the core polysaccharide , and 3 lipid A , which anchors the LPS into the outer membrane. LPS is known to serve many different functions for the cell, such as contributing to the net negative charge for the cell, helping to stabilize the outer membrane, and providing protection from certain chemical substances by physically blocking access to other parts of the cell wall.
In addition, LPS plays a role in the host response to pathogenic gram negative bacteria.
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