Bacteriocin Protein BacL1of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Abstract

Bacteriocin 41 (Bac41) is produced from clinical isolates ofEnterococcus faecalisand consists of two extracellular proteins, BacL1and BacA. We previously reported that BacL1protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycand-isoglutamyl-l-lysine endopeptidase that induces cell lysis ofE. faecaliswhen an accessory factor, BacA, is copresent. However, the target of BacL1remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of targetE. faecaliscells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1and BacA. The static growth state also abolished the binding and targeting of BacL1to the cell division-associated site. Furthermore, the targeting of BacL1was detectable among Gram-positive bacteria with anl-Ala-l-Ala-cross-bridging peptidoglycan, includingE. faecalis,Streptococcus pyogenes, orStreptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such asEnterococcus faecium,Enterococcus hirae,Staphylococcus aureus, orListeria monocytogenes. These data suggest that BacL1specifically targets thel-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses theE. faecaliscells during cell division.