RESUMEN
Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP(uv) nisin bioassay. It harbors the GFP(uv) encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP(uv) fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml(-1)) for nisin than the parent strain (10 pg ml(-1)). This showed that mutants of LAC275 could successfully be isolated using FACS.
Asunto(s)
Bioensayo , Técnicas Biosensibles , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/metabolismo , Lactococcus lactis/aislamiento & purificación , Nisina/metabolismo , FluorescenciaRESUMEN
Nisin Z, a post-translationally modified antimicrobial peptide of Lactococcus lactis, is positively autoregulated by extracellular nisin via the two-component regulatory proteins NisRK. A mutation in the nisin NisT transporter rendered L. lactis incapable of nisin secretion, and nisin accumulated inside the cells. Normally nisin is activated after secretion by the serine protease NisP in the cell wall. This study showed that when secretion of nisin was blocked, intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin. The isolated cytoplasm of a non-nisin producer could also cleave the leader from the nisin precursor, showing that the cytoplasm of L. lactis cells does contain proteolytic activity capable of cleaving the leader from fully modified nisin precursor. Nisin could not be detected in the growth supernatant of the NisT mutant strain with a nisin-sensing strain (sensitivity 10 pg ml(-1)), which has a green fluorescent protein gene connected to the nisin-inducible nisA promoter and a functional nisin signal transduction circuit. Northern analysis of the NisT mutant cells revealed that even though the cells could not secrete nisin, the nisin-inducible promoter P(nisZ) was active. In a nisB or nisC background, where nisin could not be fully modified due to the mutations in the nisin modification machinery, the unmodified or partly modified nisin precursor accumulated in the cytoplasm. This immature nisin could not induce the P(nisZ) promoter. The results suggest that when active nisin is accumulated in the cytoplasm, it can insert into the membrane and from there extrude parts of the molecule into the pseudoperiplasmic space to interact with the signal-recognition domain of the histidine kinase NisK. Potentially, signal presentation via the membrane represents a general pathway for amphiphilic signals to interact with their sensors for signal transduction.