STUDIES ON PROLYSOSTAPHIN PROCESSING AND CHARACTERIZATION OF THE LYSOSTAPHIN IMMUNITY FACTOR (LIF) OF STAPHYLOCOCCUS SIMULANS BIOVAR STAPHYLOLYTICUS

Lysostaphin is an extracellular glycylglycine endopeptidase produced b y Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses s taphylococcal cells by hydrolysing the polyglycine interpeptide bridge s of the peptidoglycan, Renewed analysis of the sequence of the lysost aphin gene (lss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal p eptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa a re organized in 15 tandem repeats of 13 aa length, and a mature protei n of 246 aa, Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine prote ase, Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active, The controlled expression in Staphylococcus carnosus of Iss and Iss with deletions in the prepro peptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss i n a less active state, Intracellularly expressed pro- and mature lysos taphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones, We characterized a lysostap hin immunity factor gene (lif) which is located in the opposite direct ion to Iss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, I ss is coexpressed the serine/glycine ratio is further increased to 58% , suggesting that Lss selects for optimal serine incorporation, Lif sh ows similarity to FemA and Feme proteins, which are involved in the bi osynthesis of the glycine interpeptide bridge of staphylococcal peptid oglycan, In contrast to that of Lif, the production of FemA and FemB i n S. carnosus does not cause lysostaphin immunity, The putative tRNASe r gene located downstream of Iss had no recognizable influence on lyso staphin immunity, Iss and lif are flanked by insertion sequences, sugg esting that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.