Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases of Staphylococcus simulans biovar staphylolyticus

Abstract Staphylococcus simulans biovar staphylolyticus secreted two bacteriolytic peptidoglycan hydrolases as proproteins that were activated as they were processed by an extracellular sulphydryl protease. This processing resulted in the production of multiple molecular-mass forms of each enzyme. Cells from early exponential phase cultures were susceptible to lysis by the mature forms of each of the peptidoglycan hydrolases whereas stationary phase cells were resistant. Thus secretion of these bacteriolytic enzymes during early exponential growth as precursors that are activated later by the protease would provide time for the cells to become resistant.     

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells had been altered by treating them with polymyxin B.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.     

Mechanism of action of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-positive bacteria: role of the cell wall anionic polymers of the target bacteria

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of teichoic and teichuronic acids, rather than on their chemical composition.     

The Mechanism of Action of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Positive Bacteria: The Role of the Cell Wall Anionic Polymers of Target Bacteria

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of the teichoic and teichuronic acids rather than on their chemical composition.     

Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system

The target range of a bacterial secretion system can be defined by effector substrate specificity or by the efficacy of effector delivery. Here, we report the crystal structure of Tse1, a type VI secretion (T6S) bacteriolytic amidase effector from Pseudomonas aeruginosa. Consistent with its role as a toxin, Tse1 has a more accessible active site than related housekeeping enzymes. The activity of Tse1 against isolated peptidoglycan shows its capacity to act broadly against Gram-negative bacteria and even certain Gram-positive species. Studies with intact cells indicate that Gram-positive bacteria can remain vulnerable to Tse1 despite cell wall modifications. However, interbacterial competition studies demonstrate that Tse1-dependent lysis is restricted to Gram-negative targets. We propose that the previously observed specificity for T6S against Gram-negative bacteria is a consequence of high local effector concentration achieved by T6S-dependent targeting to its site of action rather than inherent effector substrate specificity.     

Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan–peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an α/β hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles Δ lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan–peptidoglycan layer.     

Effects of moenomycin on Escherichia coli

The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10(-9).     

Use of a lytic enzyme system from Cytophaga sp. in the lysis of Gram-positive bacteria

A lytic enzyme system from Cytophaga sp. has been used for lysis of the Gram-positive bacteria, Bacillus and Corynebacterium. The optimum pH and temperature for the lytic reaction were 9.2 and 50°C, respectively. The effect of substrate and enzyme concentration have also been studied. Protein release was followed and the potential of using bacteriolytic enzymes for large-scale cell lysis and release of intracellular material is discussed.     

Murein transglycosylase from phage lambda lysate. Purification and properties

Lysates of induced E. coli (lambda) lysogens contain two enzymes acting on murein: endopeptidase and murein transglycosylase. The transglycosylase was separated from the endopeptidase and purified to homogeneity. Its bacteriolytic activity was 200-fold higher than of hen egg lysozyme. The bacteriolytic activity of the lysate depends on the presence of the enzyme. The endopeptidase alone not lyse the cells, but it enhances the extent of lysis. The properties of the transglycosylase (molecular weight 17 500, pH optimum at 6.6, inactivation by Zn2+), show that it is entirely different from the bacterial enzyme of the same specificity described by others. Data are presented, which suggest that this enzyme is the phage lambda R-gene product.     

On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering

Bacteriophage endolysins have the potential to be a long-term antibacterial replacement for antibiotics. The exogenous application of endolysins on some bacteria results in rapid cell lysis. The prospects for endolysins are furthered by the ability to engineer them; novel endolysins can be developed with optimised stability, specificity, and lytic function. But the success of endolysin engineering and application requires a comprehensive understanding of the relationship between the enzymes biochemical, biophysical and bacteriolytic properties. Here, we examine their catalytic mechanisms, opportunities for developing novel endolysins, and highlight areas where a better understanding would support their long-term success as antibacterial agents.     

Extraction of chromosomal DNA from Staphylococcus and Listeria by a rapid method using achromopeptidase

A rapid and simple method for preparation of chromosomal DNA from Gram-positive bacteria is reported. Susceptibility to lysis with Sodium Dodecyl Sulfate (SDS) increases when undergoing treatment with acetone before being digested by bacteriolytic enzymes. Rapid lysis of Staphylococcus and Listeria cells is obtained through a respective treatment by lysozyme with lysostaphine and by lysozyme with achromopeptidase, adding to that the effect of SDS in Tris-Hcl buffer. This procedure of preparing chromosomal DNA provides 1 to 4 mg of DNA out of 1 g of bacterial cells in a day.     

Bacteriolysis by immobilized enzymes.

Bacteriolytic enzymes produced by Achromobacter lunatus were immobilized in collagen membrane. Intact bacteria such as Pseudomonas solanacearum, Xanthomonas oryzae, Staphylococcus aureus, and Pseudomonas aeruginosa were lyzed with the bacteriolytic enzyme-collagen membrane. Relative activity of the bacteriolytic enzyme-collagen membrane against Pseu. solanacearum was about 2% of that of native bacteriolytic enzymes. No difference in the optimum pH was observed between immobilized enzymes and native enzymes. The bacteriolytic enzymes in the collagen membrane were stable against sodium chloride which was an inhibitor of the native bacteriolytic enzymes. Xanthomonas oryzae and Pseu. aeruginosa were continuously lyzed by a reactor containing the rolled bacteriolytic enzyme-collagen membrane.     

Effect of aerobic and anaerobic growth on the cell wall ofStaphylococcus aureus

Electron micrographs ofStaphylococcus aureus 7167 which had been grown anaerobically showed that the cell wall was approximately 5 times thicker than the wall of bacteria after aerobic growth. Cell walls prepared from anaerobically grownS. aureus were more sensitive to the bacteriolytic enzymes: lysostaphin, lysozyme, and the wall-associated autolytic enzyme ofB. subtilis 168 I^?. Our findings are interpreted as evidence that the cell wall or surface of anaerobically grownS. aureus 7167 is different from that of aerobically grownS. aureus 7167. The findings suggest that the cell wall peptidoglycan of the anaerobe is a more loosely formed network, resulting in a more rapid solubilization by the bacteriolytic enzymes.     

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.     

Bacterial peptidoglycan (murein) hydrolases

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and their cleavage sites is provided. The physiological functions of these enzymes include the regulation of cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis. Specialized hydrolases enlarge the pores in the peptidoglycan for the assembly of large trans-envelope complexes (pili, flagella, secretion systems), or they specifically cleave peptidoglycan during sporulation or spore germination. Moreover, peptidoglycan hydrolases are involved in lysis phenomena such as fratricide or developmental lysis occurring in bacterial populations. We will also review the current view on the regulation of autolysins and on the role of cytoplasm hydrolases in peptidoglycan recycling and induction of beta-lactamase.     

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-l-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

     

Hydrolysis of a Staphylococcus aureus cell wall peptidoglycan by 209 P lysoamidase

Hydrolysis of Staphylococcus aureus 209 P cell wall peptidoglycan was accompanied by the liberation of 1.3 mol of C-terminal and 1.2 mol of N-terminal glycine per mole of Glu as well as of 0.5 mol of N-terminal and 0.3 mol of C-terminal alanine. Gel chromatography on Sephadex G-25, ion-exchange chromatography on QAE-Sephadex A-50 and paper electrophoresis of S. aureus peptidoglycan hydrolysates gave seven homogeneous fractions; these fractions were structurally defined. Lysoamidase hydrolyzed bonds Mur-Ala, Gly-Gly and Mur-GlcN in the peptidoglycan molecule. Hydrolysis of glycan chains was accompanied by the formation of large fragments, (GlcN-Mur)9 and (GlcN-Mur)28. The lytic effect of lysoamidase on S. aureus peptidoglycan is coupled with bacteriolytic enzymes of lysoamidase: acetmuramyl amidase, glycyl--glycine endopeptidase and acetyl--muramidase.     

Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors

Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.