Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

Abstract

Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH 2 SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH 2 SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.Most eubacteria are mechanically stabilized by the shapedetermining peptidoglycan, which consists of polysaccharide chains that are connected by short peptides. Expansion of the bacterial cell wall during growth and splitting of the septum prior to cell separation require enzymes that cleave some of the existing covalent bonds within the peptidoglycan sacculus. These enzymes are collectively known as peptidoglycan hydrolases (24). Some have been characterized as N-acetylmuramidases, N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, and transglycosidases (10). Most peptidoglycan hydrolases have been characterized as bacteriolytic enzymes by in vitro studies (29). Lysostaphin, which is active against Staphylococcus aureus, is one such enzyme. It has been suggested that lysostaphin may function catabolically to release nutrients from other staphylococci in the environment, with the producer cell being protected by a specific immunity protein (12,18). The lytic effect of lysostaphin results from a direct attack on the integrity of the staphylococcal cell wall, specifically bringing about cleavage of the pentaglycine crosslink between the stem peptides of individual peptidoglycan subunits (4).Zoocin A, a bacteriolytic cell wall hydrolase produced by Streptococcus zooepidemicus 4881, like lysostaphin, cleaves bonds within the peptide moiety of peptidoglycan (26). Zoocin A has been shown to inhibit the growth of all Streptococcus pyogenes strains and all S. zooepidemicus strains other than 4881 itself (14). Both enzymes, lysostaphin and zoocin A, possess a catalytic domain in the N-terminal portion and a...

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“…Peptidoglycan (1 mg/ml) was incubated with millericin B (10 g/ml) and aliquots (10 to 50 nmol, estimated from a standard curve) taken at different time intervals to determine the liberation of free amino groups (6). Flourodinitrobenzene (FDNB) reagent (130 l in 10 ml of 100% ethanol) was added to each aliquot, mixed, and incubated at 60°C for 30 min.…”

“…Millericin B, a peptidoglycan hydrolase produced by Streptococcus milleri NMSCC061, was purified and partially characterized as an endopeptidase that cleaves the interpeptide cross bridge and the stem peptide of susceptible peptidoglycan (6). This suggests that millericin B has activity similar to that of lysostaphin and ALE-1, both of which are glycylglycine endopeptidases that are produced by Staphylococcus simulans bv.…”