Self-protection against cell wall hydrolysis in Streptococcus milleri NMSCC 061 and analysis of the millericin B operon

Streptococcus milleri NMSCC 061 produces an endopeptidase, millericin B, wh ich hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. T he nucleotide sequence of a 4.9-kb chromosomal region showed three open rea ding frames (ORFs) and a putative tRNA(Leu) sequence. The three ORFs encode a millericin B preprotein (MiIB), a putative immunity protein (MiIf), and a putative transporter protein (MiIT). The milB gene encodes a 277-amino-ac id preprotein with an 18-amino-acid signal peptide with a consensus IIGG cl eavage motif. The predicted protein encoded by milT is homologous to ABC (A TP-binding cassette) transporters of several bacteriocin systems and to pro teins implicated in the signal-sequence-independent export of Escherichia c oli hemolysin A. These similarities strongly suggest that the milT gene pro duct is involved in the translocation of millericin B. The gene milF encode s a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of Staphylococcus aureus, which are involved in the addition of g lycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell w all mucopeptide of S. milleri NMSCC 061(resistant to lysis by millericin B) and S. milleri NMSCC 051 (sensitive) showed a single amino acid difference . Serial growth of S. milleri NMSCC 051 in a cell wall minimal medium conta ining an increased concentration of leucine resulted in the in vivo substit ution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of S. milleri NMSCC 051 (sensitive) that contained an amino ac id substitution (leucine for threonine) within its peptidoglycan cross brid ge showed partial susceptibility to millericin B. The putative tRNA(Leu) se quence located upstream of milB may be a cell wall-specific tRNA and could together with the milF protein, play a potential role in the addition of le ucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.