Zoocin A immunity factor: a femA-like gene found in a group C streptococcus

A 6.8-kb fragment of Streptococcus equi subsp. zooepidemicus 4881 DNA containing the zoocin A gene (zooA) was cloned in Escherichia coli and sequenced. We have identified a gene we call zoocin A immunity factor (zif), which protects the producer cell from the otherwise lethal action of its own product. Transformation of Streptococcus gordonii DL1 with zooA and zif changed its phenotypic character from a non-zoocin A producing-zoocin A sensitive cell to a zoocin A producing-zoocin A resistant cell. zif has sequence homology to femA (factor essential for methicillin resistance) and lif (lysostaphin immunity factor). No differences were observed in amino acid or amino sugar compositions of peptidoglycan purified from zoocin A sensitive vs. zoocin A immune cells.     

Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.     

The streptococcolytic enzyme zoocin A is a penicillin-binding protein

Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that has an unknown site of action on the peptidoglycans of susceptible organisms. Analysis of a mutant strain in which the genes for zoocin A and resistance to zoocin A were inactivated revealed that this strain was more susceptible to beta-lactam antibiotics than the parental organism. Purified zoocin A had weak beta-lactamase activity, bound radioactive penicillin covalently, and its streptococcolytic activity was inhibited by penicillin. Thus, zoocin A is a penicillin-binding protein and presumably is a D-alanyl endopeptidase.     

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

Cloning and sequence analysis of zooA,a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin

The nucleotide sequence has been determined for zooA, a gene encoding the bacteriocin-like inhibitory substance zoocin A in Streptococcus zooepidemicus strain 4881. The zooA gene product corresponds to the 285-amino acid (aa) zoocin A pre-peptide from which a leader sequence is cleaved to form the 262-aa biologically active molecule of estimated molecular mass 27 877 Da. Expression of zooA in a Gram-negative host was shown by the extracellular release from Escherichia coli, containing cloned zooA, of a biologically active peptide having an identical range of anti-bacterial activity to that of zoocin A, purified from S. zooepidemicus strain 4881. Data base searches revealed sequences having homologies with known muralytic proteins produced by both Gram-positive and Gram-negative bacteria and indicate a `mix and match' blending of domain-type structures, the C-terminal putative receptor-recognition region of the molecule being joined by a threonine-proline-rich linker to an N-terminal putative catalytic region having homology with several known endopeptidases, including lysostaphin.     

Solution structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain of Zoocin A

Zoocin A is a Zn‐metallopeptidase secreted by Streptococcus zooepidemicus strain 4881. Its catalytic domain is responsible for cleaving the D‐alanyl‐L‐alanine peptide bond in streptococcal peptidoglycan. The solution NMR structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain (rCAT C74A) has been determined. With a previous structure determination for the recombinant target recognition domain (rTRD), this completes the 3D structure of zoocin A. While the structure of rCAT C74A resembles those of the catalytic domains of lysostaphin and LytM, the substrate binding groove is wider and no tyrosine residue was observed in the active site. Proteins 2016; 85:177–181. © 2016 Wiley Periodicals, Inc.     

In Vitro Characterization of Peptidoglycan-Associated Lipoprotein (PAL)–Peptidoglycan and PAL–TolB Interactions

The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.     

Lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors

Empedopeptin is a natural lipodepsipeptide antibiotic with potent antibacterial activity against multiresistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae in vitro and in animal models of bacterial infection. Here, we describe its so far elusive mechanism of antibacterial action. Empedopeptin selectively interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes and their respective purified substrates, we show that empedopeptin forms complexes with undecaprenyl pyrophosphate containing peptidoglycan precursors. The primary physiological target of empedopeptin is undecaprenyl pyrophosphate-N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Lipid II is bound in a region that involves at least the pyrophosphate group, the first sugar, and the proximal parts of stem peptide and undecaprenyl chain. Undecaprenyl pyrophosphate and also teichoic acid precursors are bound with lower affinity and constitute additional targets. Calcium ions are crucial for the antibacterial activity of empedopeptin as they promote stronger interaction with its targets and with negatively charged phospholipids in the membrane. Based on the high structural similarity of empedopeptin to the tripropeptins and plusbacins, we propose this mechanism of action for the whole compound class.     

Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.     

LambdaSa1 and LambdaSa2 prophage lysins of Streptococcus agalactiae

Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity.     

The metal binding site of zoocin A

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site.On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2 +/- 0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.     

Specificity of the autolysin of Streptococcus (Diplococcus) pneumoniae

A Streptococcus (Diplococcus) pneumoniae autolysin, partially purified from cellular autolysates, was optimally active at pH 7.0 and was stimulated by monovalent cations. Addition of autolysin to walls resulted in the appearance of only N-terminal l-alanine, whereas no glycosidase activity was observed. Walls which had been solubilized by autolysin were separated by gel filtration into a low-molecular-weight peptide containing amino acids in the same ratios found in intact walls and a high molecular fraction containing the amino acid-deficient peptidoglycan backbone. Thus, the major activity is an N-acetylmuramyl-l-alanine amidase. In addition, walls undergoing spontaneous lysis revealed no glycosidase activity but showed an increase in only N-terminal alanine. Autolysin, which was bound to walls in saline, was almost completely removed when walls were washed in distilled water, and all of the activity was recovered in the water wash fluid.     

Diversity of bacteriocins in the microbiome of the Tucuruí Hydroelectric Power Plant water reservoir and three-dimensional structure prediction of a zoocin

Bacteriocins are antimicrobial peptides expressed by bacteria through ribosomal activity. In this study, we analyzed the diversity of bacteriocin-like genes in the Tucuruí-HPP using a whole-metagenome shotgun sequencing approach. Three layers of the water column were analyzed (photic, aphotic and sediment). Detection of bacteriocin-like genes was performed with blastx using the BAGEL4 database as subject sequences. In order to calculate the abundance of bacteriocin-like genes we also determined the number of 16S rRNA genes using blastn. Taxonomic analysis was performed using RAST server and the metagenome was assembled using IDBA-UD in order to recover the full sequence of a zoocin which had its three-dimensional structure determined. The photic zone presented the highest number of reads affiliated to bacteriocins. The most abundant bacteriocins were sonorensin, Klebicin D , pyocin and colicin. The zoocin model was composed of eight anti-parallel β-sheets and two α-helices with a Zn²⁺ ion in the active site. This model was considerably stable during 10 ns of molecular dynamics simulation. We observed a high diversity of bacteriocins in the Tucuruí-HPP, demonstrating that the environment is an inexhaustible source for prospecting these molecules. Finally, the zoocin model can be used for further studies of substrate binding and molecular mechanisms involving peptidoglycan degradation.     

Proposed docking interface between peptidoglycan and the target recognition domain of zoocin A

A docking model is proposed for the target recognition domain of the lytic exoenzyme zoocin A with the peptidoglycan on the outer cell surface of sensitive bacterial strains. Solubilized fragments from such peptidoglycans perturb specific backbone and side chain amide resonances in the recombinant form of the domain designated rTRD as detected in two-dimensional ¹H–¹⁵N correlation NMR spectra. The affected residues comprise a shallow surface cleft on the protein surface near W115, N53, N117, and Q105 among others, which interacts with the peptide portion of the peptidoglycan. Calculations with AutoDock Vina provide models of the docking interface. There is approximate homology between the rTDR-peptidoglycan docking site and the antigen binding site of Fab antibodies with the immunoglobin fold. EDTA was also found to bind to rTRD, but at a site distinct from the proposed peptidoglycan docking site.     

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. II. Immunological mechanisms involved in thrombocytolysis.

Immunological mechanisms are involved in the thrombocytolytic activity of peptidoglycan of Group A streptococcus, Streptococcus pneumoniae and Staphylococcus aureus. Inactivation of particular components of complement (heating of blood serum to 56 degrees C,incubation with zymosan or NH4OH) inhibited the thrombocytolytic activity of group A streptococcus peptidoglycan. So did preincubation of Group A streptococcus peptidoglycan with homologous antipeptidoglycan antibody. On the other hand, antibody to Group A streptococcus peptidoglycan did not inhibit the thrombocytolytic effect of Streptococcus pneumoniae or Staphylococcus aureus peptidoglycan. Human platelets are resistant to peptidoglycans. They remain resistant in the presence of rabbit serum although rabbit platelets are highly sensitive to peptidoglycans. This suggests that, for the expression of the thrombocytolytic activity of bacterial peptidoglycan, specific receptors on the surface of platelets must be present in addition to serum factors.     

Occurrence of Glucosamine Residues with Free Amino Groups in Cell Wall Peptidoglycan from Bacilli as a Factor Responsible for Resistance to Lysozyme

Analysis by dinitrophenylation techniques revealed the occurrence of significant amounts of glucosamine residues with free amino groups in the peptidoglycan component of cell walls isolated from Bacillus cereus, Bacillus subtilis, and Bacillus megaterium. A close correlation was demonstrated between the content of N-unacetylated glucosamine residues in the peptidoglycan component and the resistance of the cell walls to lysozyme. These lysozyme-resistant cell walls and peptidoglycan were converted into a lysozyme-sensitive form by means of N-acetylation with acetic anhydride. Thus, the occurrence of the N-unacetylated glucosamine residues in the peptidoglycan component accounts for the resistance of these cell walls to lysozyme. The N-unacetylated glucosamine residues were not found in a significant amount in the cell walls of Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus faecalis, Lactobacillus casei, or Lactobacillus arabinosus.     

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.