The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1574-6976.2007.00094.x

Peptidoglycan structure and architecture

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

It is clear that the above can lead to confusion when scientists of different countries are trying to communicate with each other. Another example is the burrowing rodent called a gopher found throughout the western United States. In the southeastern United States the term gopher refers to a burrowing turtle very similar to the desert tortoise found in the American southwest. One final example; two North American mammals known as the elk and the caribou are known in Europe as the reindeer and the elk. We never sing “Rudolph the Red-nosed elk”! Confused? This was the reason for creating an internationally recognized system of naming organisms. To avoid confusion, living organisms are assigned a scientific name based on Latin or Latinized words. The English sparrow is Passer domesticus or Passer domesticus (italics or underlining these two names is the official written representation of a scientific name). Using a uniform naming system allows scientists from all over the world to recognize exactly which life form a scientist is referring to. The naming process is called the binomial system of nomenclature. Passer is comparable to a surname and is called the genus, while domesticus is the specific or species name (like your given name) of the English sparrow. Now scientists can give all sparrow-like birds the genus Passer but the species name will vary. All similar genera (plural for genus) can be grouped into another, “higher” category (see below). Study the following for a more through understanding of taxonomy. Taxonomy Analogy Kingdom: Animalia Country

Diversity of life

Summary: Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a “second generation” of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases.

Three Decades of β-Lactamase Inhibitors

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.

Ligation-independent cloning of PCR products (LIC-PCR)

The monofunctional penicillin-binding DD-peptidases and penicillin-hydrolyzing serine beta-lactamases diverged from a common ancestor by the acquisition of structural changes in the polypeptide chain while retaining the same folding, three-motif amino acid sequence signature, serine-assisted catalytic mechanism, and active-site topology. Fusion events gave rise to multimodular penicillin-binding proteins (PBPs). The acyl serine transferase penicillin-binding (PB) module possesses the three active-site defining motifs of the superfamily; it is linked to the carboxy end of a non-penicillin-binding (n-PB) module through a conserved fusion site; the two modules form a single polypeptide chain which folds on the exterior of the plasma membrane and is anchored by a transmembrane spanner; and the full-size PBPs cluster into two classes, A and B. In the class A PBPs, the n-PB modules are a continuum of diverging sequences; they possess a five-motif amino acid sequence signature, and conserved dicarboxylic amino acid residues are probably elements of the glycosyl transferase catalytic center. The PB modules fall into five subclasses: A1 and A2 in gram-negative bacteria and A3, A4, and A5 in gram-positive bacteria. The full-size class A PBPs combine the required enzymatic activities for peptidoglycan assembly from lipid-transported disaccharide-peptide units and almost certainly prescribe different, PB-module specific traits in peptidoglycan cross-linking. In the class B PBPs, the PB and n-PB modules cluster in a concerted manner. A PB module of subclass B2 or B3 is linked to an n-PB module of subclass B2 or B3 in gram-negative bacteria, and a PB module of subclass B1, B4, or B5 is linked to an n-PB module of subclass B1, B4, or B5 in gram-positive bacteria. Class B PBPs are involved in cell morphogenesis. The three motifs borne by the n-PB modules are probably sites for module-module interaction and the polypeptide stretches which extend between motifs 1 and 2 are sites for protein-protein interaction. The full-size class B PBPs are an assortment of orthologs and paralogs, which prescribe traits as complex as wall expansion and septum formation. PBPs of subclass B1 are unique to gram-positive bacteria. They are not essential, but they represent an important mechanism of resistance to penicillin among the enterococci and staphylococci. Natural evolution and PBP- and beta-lactamase-mediated resistance show that the ability of the catalytic centers to adapt their properties to new situations is limitless. Studies of the reaction pathways by using the methods of quantum chemistry suggest that resistance to penicillin is a road of no return.

Multimodular Penicillin-Binding Proteins: An Enigmatic Family of Orthologs and Paralogs

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-α and α/β folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK d,d-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4→3) type. They are inactivated by penicillin and other β-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4→3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze d,d peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine β-lactamases, and the penicillin sensors of several penicillin sensory transducers help the d,d-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Penr protein fusions. Plausible hypotheses are put forward on the roles that the Penr protein fusions, acting as l,d-acyltransferases, may play in the (3→3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4→3) type to the (3→3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

Intracellular bacterial pathogens enter their hosts surrounded by a membrane-bound vacuole and use a panel of tricks to exploit or evade eukaryotic cell functions ([9][1], [12][2]). Chlamydia inhabits vesicles that do not fuse with lysosomes and remains within these parasitophorous vacuoles (termed

Lack of Cell Wall Peptidoglycan Versus Penicillin Sensitivity: New Insights into the Chlamydial Anomaly

Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.

Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase

The ftsI-encoded multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation machinery of Escherichia coli. Altered ftsI genes were overexpressed, and the gene products were analyzed with respect to the level of production, stability, penicillin affinity, and cell septation activity. In contrast to the serine beta-lactamases and low-molecular-mass PBPs which are autonomous folding entities, the S-259-to-V-577 penicillin-binding module of M-1-to-V-577 PBP3 lacks the amino acid sequence information for correct folding. The missing piece of information is provided by the associated G-57-to-E-258 non-penicillin-binding module which functions as a noncleaved, pseudointramolecular chaperone. Key elements of the folding information reside within the motif 1-containing R-60-to-W-110 polypeptide segment and within G-188-to-D-197 motif 3 of the n-PB module. The intermodule interaction is discussed in the light of the known three-dimensional structure (at 3.5-A [0.35-nm] resolution) of the analogous class B PBP2x of Streptococcus pneumoniae (S. Pares, N. Mouz, Y. Petillot, R. Hakenbeck, and O. Dideberg, Nature Struct. Biol. 3:284-289, 1996). Correct folding and adoption of a stable penicillin-binding conformation are necessary but not sufficient to confer cell septation activity to PBP3 in exponentially growing cells. The in vivo activity of PBP3 also depends on the M-1-to-E-56 amino-terminal module which encompasses the cytosol, the membrane, and the periplasm and which functions as a noncleaved pseudo-signal peptide.

The non-penicillin-binding module of the tripartite penicillin-binding protein 3 of Escherichia coli is required for folding and/or stability of the penicillin-binding module and the membrane-anchoring module confers cell septation activity on the folded structure.

Colette Goffin

Papers

Multimodular Penicillin-Binding Proteins: An Enigmatic Family of Orthologs and Paralogs

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

Lack of Cell Wall Peptidoglycan Versus Penicillin Sensitivity: New Insights into the Chlamydial Anomaly

Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase

The non-penicillin-binding module of the tripartite penicillin-binding protein 3 of Escherichia coli is required for folding and/or stability of the penicillin-binding module and the membrane-anchoring module confers cell septation activity on the folded structure.