Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50▿

摘要

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.