Self-Protection against Cell Wall Hydrolysis in Streptococcus milleri NMSCC 061 and Analysis of the Millericin B Operon

Abstract

Several bacterial species produce peptidoglycan hydrolases that lyse target cells (4,8,10,33,43). The physiological functions of these enzymes remain largely unknown. The suggestion that bacteria use them to gain a competitive advantage in environments where nutrients are limited has been made previously (49). They may also allow producer organisms to establish an ecological niche within a competitive environment (49).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. staphylolyticus and Staphylococcus capitis EPK1, respectively (43, 47), as well as zoocin A, an endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 (4).Molecular cloning of the ale-1 gene showed that the primary structure of the mature ALE-1 is very similar to that of the proenzyme form of lysostaphin. The genes for lysostaphin (end) in S. simulans bv. staphylolyticus and for ALE-1 (ale-1) in S. capitis EPK1 were found to be located on large plasmids (20,22,35). This was in contrast to a previous finding where the gene for lysostaphin was found to be located on the chromosome of S. simulans bv. staphylolyticus (24).S. simulans bv. staphylolyticus is resistant to lysis by lysostaphin (37), and this resistance is conferred by modifying the amino acid composition of interpeptide chains in cell wall peptidoglycan by increasing the serine content and decreasing the glycine content (11). The genetic elements for production and self-protection (immunity) of both lysostaphin and ALE-1 have been characterized (11,22,47,50). The gene involved in the modification of peptidoglycan (epr, for endopeptidase resistance) was shown to be located on a large plasmid, pACK1, together with the gene (end) which encodes the endopeptidase (22). Staphylococcus aureus cells transformed with a plasmid containing the 8.4-kb DNA fragment from pACK1 produced lysostaphin and were resistant to lysis by lysostaphin, which indicated that the DNA fragment contained both epr and end (11).The objective of this study was to determine whether the genes for millericin B and millericin B host immunity in S. milleri NMSCC 061 are similar in organization to the genes for lysostaphin and lysostaphin host immunity in S. simulans bv. staphylolyticus and the genes for ALE-1 and ALE-1 host immunity in S. capitis EPK1 (46,47,50). Here we describe the sequencing of the millericin B gene (milB) and the identification and sequencing of three additional genes in S. milleri NMSCC 061 that appear to be associated with millericin B host self-protection and its export. One of these genes (milF, for millericin B immunity factor), which is similar in sequence to genes that have been implicated in the synthesis of peptido...

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

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