Nisin is a 34-residue antibacterial peptide produced by Lactococcus lactis that is active against a wide range of gram-positive bacteria. In non-nisin-producing L. lactis, nisin resistance could be conferred by a specific nisin resistance gene (nsr), which encodes a 35-kDa nisin resistance protein (NSR). However, the mechanism underlying NSR-mediated nisin resistance is poorly understood. Here we demonstrated that the protein without the predicted N-terminal signal peptide sequence, i.e., NSRSD, could proteolytically inactivate nisin in vitro by removing six amino acids from the carboxyl "tail" of nisin. The truncated nisin (nisin 1-28 ) displayed a markedly reduced affinity for the cell membrane and showed significantly diminished pore-forming potency in the membrane. A 100-fold reduction of bactericidal activity was detected for nisin 1-28 in comparison to that for the intact nisin. In vivo analysis indicated that NSR localized on the cell membrane and endowed host strains with nisin resistance by degrading nisin as NSRSD did in vitro, whereas NSRSD failed to confer resistance upon the host strain. In conclusion, we showed that NSR is a nisin-degrading protease. This NSR-mediated proteolytic cleavage represents a novel mechanism for nisin resistance in non-nisin-producing L. lactis.

An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.