Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: Detection by specific peptide-directed antibodies

Antibodies against enterocin A were obtained by immunization of rabbits wit h synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-te rminal amino acid sequences of enterocin A and conjugated to the carrier pr otein KLH, Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only rea cted with enterocin A because the amino acid sequences of the C-terminal pa rts of class IIa bacteriocins are highly variable. Enterocin A and/or pedio cin PA-1 structural and immunity genes were introduced in Lactococcus lacti s IL1403 to achieve (co)production of the bacteriocins. The level of produc tion of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacte riocin translocation machinery of IL1403. Despite the low production levels , both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the a nti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J, M, Mar tinet, M. I. Martinet, A. M, Suarez, C. Herranz, P, Casaus, L, M, Cintas, J , M. Rodriguez, and P. E, Hernandez, Appl, Environ. Microbiol. 64:4536-4545 , 1998), In this work the availability of antibodies for the specific detec tion and quantification of enterocin A and pediocin PA-1 was crucial to dem onstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), beca use indicator strains that are selectively inhibited by each bacteriocin ar e not available.