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Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS) are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci.

The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map individual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.

Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules.

AcmA, the major autolysin of Lactococcus lactis MG1363, is responsible for stationary phase cellular lysis and is involved in cell separation of this organism (9). The enzyme consists of two domains: the N-terminal region contains an N-acetyl-glucosaminidase active site domain (9; A. Steen, G. Buist, G. Horsburgh, S. J. Foster, O. P. Kuipers, and J. Kok, unpublished data) while the C-terminal region contains three so-called LysM domains, with which it specifically binds to peptidoglycan of L. lactis and of other gram-positive bacteria (49). Peptidoglycan, the major cell wall component in bacteria and the substrate of AcmA, consists of glycan strands cross-linked by peptide side chains. The peptide chain contains alternating L-and D-amino acids. D-Alanine (D-Ala) is incorporated into the peptidoglycan peptide moiety as a D-Ala-D-Ala dipeptide, where it is involved in cross-linking of adjacent peptidoglycan strands. In many bacteria alanine racemase is responsible for the synthesis of D-Ala from L-Ala, the naturally occurring alanine isomer (53). Bacillus subtilis expresses at least one alanine racemase: Dal (14). A dal mutant is dependent on D-Ala supplementation to be able to grow in a rich medium; cells start to lyse in the absence of D-Ala (4,14,20). In minimal medium the mutant is D-Ala dependent when L-Ala is supplemented, suggesting that a second, L-Ala-repressible racemase is present (4,14). Lactobacillus plantarum probably expresses only one alanine racemase, as an alr mutant is totally dependent on D-Ala for growth (22). D-Ala deprivation of an alr mutant of L. plantarum resulted in growth arrest, a rapid loss of cell viability, and an aberrant cell morphology (43). Electron microscopy analyses showed that mainly the cell septum is affected in this mutant. Like L. plantarum alr, L. lactis alr is totally dependent on the addition of D-Ala to the growth medium (23); when D-Ala was removed from the growth medium when the cells were in exponential growth phase, L. lactis alr growth was impaired and the culture started to lyse (17). The alr gene was used as a food-grade plasmid selection marker in the alr mutants of L. plantarum and L. lactis, complementing the D-Ala auxotrophy (7). Moreover, the alr mutants of L. plantarum and L. lactis were used in a mucosal vaccination study, in which these two mutants were shown to enhance the mucosal delivery of the tetanus toxin fragment C model antigen in mice (17).Although peptidoglycan covers the whole surface of L. lactis, AcmA binds to lactococcal cells at specific loci, namely around the poles and septum of the cell, exactly those places where cell lysis has been shown to start (35,49). Trichloroacetic acid treatment of cells causes binding of AcmA over the whole cell surface. Lipoteichoic acid (LTA) is a candidate-hindering component that is removed by this treatment, as it seems to be present in L. lactis at those positions where AcmA is not able to bind (49). LTA is a secondary cell wall polymer suggested to be involved in the control of autolysin activity (5, ...

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.Teichoic acids (TAs) are essential polymers found in grampositive bacteria and represent up to 50% of the cell wall dry weight (17). Lactobacillus plantarum contains two types of TAs: lipoteichoic acids (LTA) and wall teichoic acids (WTA). The L. plantarum LTA are polyglycerophosphate polymers anchored in the membrane through a glycolipid. They are highly substituted with D-alanyl esters (D-Ala:phosphate [P] ratio of 0.89) and to a minor extent with glucose (Glc:P ratio of 0.11) (2). L. plantarum WTA are polyribitolphosphates covalently bound to the peptidoglycan via a linkage unit (22). They also carry D-Ala and Glc residues but in strain-dependent variable ratios (14).Despite their essentiality, our understanding of the physiological role(s) of TAs is still incomplete (17). D-Alanyl substitutions strongly contribute to the function of TAs, and the study of mutants deficient in D-alanylation is of interest to expand our insight into the physiological role of these substitutents. The positively charged amino groups of D-alanyl esters partially counteract the negative charges of the backbone phosphate groups. Consequently, D-alanyl esters can modulate cell envelope properties and the function of several extracellular proteins (for a review, see reference 24). The biosynthesis of D-alanyl-LTA was extensively studied with Lactobacillus. rhamnosus (formerly casei) (10,18,19,24,25) and with Bacillus subtilis (27). D-Alanylation requires four proteins (DltA/Dcl, DltB, DltC/Dcp, and DltD) encoded in the dlt operon. In B. subtilis, which contains both LTA and WTA, inactivation of dlt genes prevents D-alanylation of both types of TAs (27). The dlt mutants exhibit an extended variety of phenotypes (for a review, see reference 24). For L. plantarum, it was recently demonstrated that the dlt mutant analyzed in this work displayed an enhanced anti-inflammatory capacity in a murine model of colitis (16). A large number of phenotypes can be associated with the higher-density of negative charges in the cell wall resulting from the lack of D-alanyl esters (for a review, see reference 24). As an example, TAs are thought to be involved in the control of autolysins through electrostatic interactions (12,29). Autolysins play an important role in autolysis, cell separation, and peptidoglycan...

. 178:5431-5437, 1996). Production of D-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for D-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires L-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by L-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of L-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is D-lactate dependent. We also show that the growth defect can be partially restored by expression of the D-alanyl-D-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various D-2-hydroxy acids but not D-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in D-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in D-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for D-lactate production upon inactivation or inhibition of the LdhD enzyme.In lactic acid bacteria (LAB), the pyruvate formed by the Embden-Meyerhof-Parnas pathway is reduced to lactate by NAD-dependent lactate dehydrogenases (Ldh). These enzymes are stereospecific and produce D-lactate (LdhD, EC 1.1.1.28) or L-lactate (LdhL, EC 1.1.1.27). LAB can be classified on the basis of the lactate stereoisomer(s) produced during growth on glucose, which is thought to reflect the type of Ldh(s) present in a species. LAB are usually divided in three groups based on the ratio of isomers produced (21,31,39,45).Most lactobacilli are DL-lactate producers, but the ratio of the two isomers is highly variable. This has mainly been attributed to different activities of the LdhD and LdhL enzymes (21, 45). Some exceptions among lactobacilli are Lactobacillus delbrueckii subsp. bulgaricus, which produces mainly D-lactate, in agreement with the absence of LdhL activity, and Lactobacillus casei, where L-lactate is the major isomer formed (21,39). In this species, the pathway of D-lactate production has not been investigated.The presence of a lactate racemase (EC 5. (27), and several halophilic archaea (41). Very few biochemical studies on lactate racemase have been reported. This is mainly due to the fact that the enzyme seems to be highly sensitive to oxidation (13,45). The enzymes from L. sakei and C. beijerinckii have been purified, and basic biochemical properties have been determined (9,29). A catalytic mechanism has been proposed for the lactate racemase of...

The cell wall of lactic acid bacteria has the typical gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attributes of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.