Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q

Enterococcus faecium L50 grown at 16 to 32 degreesC produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-li ke peptides synthesized without an N-terminal leader sequence or signal pep tide. However, the bacteriocin activity found in the cell-free culture supe rnatants following growth at higher temperatures (37 to 47 degreesC) is not due to EntL50, A purification procedure including cation-exchange, hydroph obic interaction, and reverse-phase liquid chromatography has shown that th e antimicrobial activity is due to two different bacteriocins. Amino acid s equences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produ ced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Havarstein, P. E. Her nandez, and I. F. Nes. Appl. Environ. Microbiol. 63: 4321-4330, 1997) and t he other is a novel unmodified non-pediocin-like bacteriocin termed enteroc in Q (EntQ), with a molecular mass of 3,980, DNA sequencing analysis of a 9 63-bp region of E, faecium L50 containing the enterocin P structural gene ( entP) and the putative immunity protein gene (entiP) reveals a genetic orga nization identical to that previously found in E. faecium P13, DNA sequenci ng analysis of a 1,448-bp region identified two consecutive but diverging o pen reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-aci d protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was Located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E, faecium L50 show ed that EntP and EntQ are produced in the temperature range from 16 to 47 d egreesC and maximally detected at 47 and 37 to 47 degreesC, respectively, w hile EntL50A and EntL50B are maximally synthesized at 16 to 25 degreesC and are not detected at 37 degreesC or above.