Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases ofStaphylococcus simulansbiovarstaphylolyticus

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.

Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

“…Alternatively, GN I and II may be the product ofa single gene and subsequently modified by proteinases present in the culture filtrate. This type of proteolytic processing has been reported previously with the production of multiple molecular-mass forms of peptidoglycan hydrolases from a single proprotein of Staphylococcus simulans [55]. However, the observed differences in physico-chemical properties of the A. persicinum glucanases, such as pH and temperature optima and stability, kinetic parameters, effects of metal ions and inhibitors and pl values, all suggest that they are separate distinct enzymes and not artefacts of proteolytic activity.…”

“…Some differences in the thermal stability of the purified f8-glucanases were also observed ( Figure 5). GN I appeared relatively stable at 55 'C, retaining more than 90 % of its activity after 30 min. At 60 'C, however, rapid inactivation occurred, with less than 50 % Temperature (IC) Figure 5 Effects of pH and temperature on the purffled A. persicinum GNs 0, pH and temperature optima; 0, pHand temperature-stability.…”

A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization.

“…Since the cloned DNA fragment of pAT4 encoded the C-terminal part of AM and the GL protein in the same polypeptide, the results indicate that the C-terminal part of AM and the entire GL protein were translated in a single polypeptide chain in this clone. Since AM and GL were purified from culture broth as separate enzymes, the bifunctional autolysin encoded by atl must be processed to generate the two extracellular cell wall hydrolases, perhaps in a manner described for the peptydoglycan hydrolases of Staphylococcus simulans (26).…”

Lysostaphin: Engineering and Potentiation toward Better Applications

“…Lysostaphin is naturally secreted by Staphylococcus simulans biovar staphylolyticus ,, when the availability of carbon sources is insufficient, and the encoding gene is present on plasmid pACK1 of this bacterium − using UUG as a start codon . The enzyme is produced as a preproprotein with 493 amino acids in the late-exponential phase, which is then secreted via an N-terminal signal peptide (36 amino acids) to become a proprotein with 15 tandem repeats of 13 amino acids located at the N-terminus. , The pro-sequence (211 amino acids) is then cleaved by a genome-encoded autoactivating extracellular cysteine protease , to release the mature enzyme (246 amino acids) in the stationary phase . Lysostaphin is dispensable for the growth of the producer cells, which encode a resistance factor named Epr (endopeptidase resistance) or Lif (lysostaphin immunity factor) to avoid autolysis. , Epr modifies the cell wall of S. simulans biovar staphylolyticus by replacing the pentaglycine crossbridge with GGSGG or GGGGS. , The epr gene is located on plasmid pACK1 adjacent to the lysostaphin gene in an opposite orientation …”

“…6 The enzyme is produced as a preproprotein with 493 amino acids in the late-exponential phase, which is then secreted via an Nterminal signal peptide (36 amino acids) to become a proprotein with 15 tandem repeats of 13 amino acids located at the N-terminus. 6,9 The pro-sequence (211 amino acids) is then cleaved by a genome-encoded autoactivating extracellular cysteine protease 10,11 to release the mature enzyme (246 amino acids) 12 in the stationary phase. 4 Lysostaphin is dispensable for the growth of the producer cells, which encode a resistance factor named Epr (endopeptidase resistance) or Lif (lysostaphin immunity factor) to avoid autolysis.…”