| Nisin is a peptide bacteriocin with highly post-translational modification, which is produced by some Lactococcus lactis strains which belong to the N serotype. Nisin as a non-toxic to the human body's natural preservative is widely applied in the food industry, paper making, agricultural feed, reproduction and contraception, medical implants and other fields.Nisin is known to exert a high antimicrobial activity against Gram-positive bacteria by four ways:inhibition of cell wall synthesis; the formation of trans-membrane pore leading to leakage of intracellular small molecules; at high concentrations nisin releases autolytic enzymes in the cell wall needed for cell division; and inhibition of spore formation. In order to avoid the self-destruction, the nisin-producing L. lactis strains protect themselves against the bactericidal activity of nisin through synergy of immunity proteins NisI and NisFEG encoded by the nisin gene cluster. In recent years some studies have shown that non-nisin-producing L. lactis strains can develop nisin resistance under certain circumstance, indicating that nisin immunity/resistance system is a complex regulatory network. In addition to nisin gene cluster, there are many genes involved in the regulation of nisin resistance in Lactococcus lactis.In this study, using mini-Mu transposition technique in nisin-producing L. lactis N8, we generated genomic random insertion mutant library of approximately 1,800 member clones. A mutant strain with improved nisin resistance was isolated and named L31. Through gene cloning, sequencing and genetic location analysis, the genes inserted by Mu transposon DNA were determined as irpT of unknown function.We studied the biological functions of irpT gene to explore its role in the regulatory networks involved in nisin resistance. First, southern blotting was performed, and it demonstrated the L. lactis L31 had only one Mu transposon DNA copy in the genomic DNA. Sequence analysis of homologous gene and genetic complementation analysis excluded the possibility of polar effects. Comparative analysis of irpT gene sequence and its encoded protein motifs, the results show that it is a trans-membrane protein, may be related to antibiotic resistance in bacteria. Western blot analysis showed that the disruption of irpT did not change the levels of the lipoprotein NisI and proteins NisFEG. Two-dimensional gel electrophoresis results showed that the disruption of irpT is caused by the significant changes of five protein spots. Phosphocarrier protein Hpr expression was significantly reduced, and the expressions of dTDP-L-rhamnose synthase, phosphopyruvate hydratase, glyceraldehyde 3-phosphate dehydrogenase and an unknown function protein were significantly increased. The transcriptional analysis confirmed up-and down-regulation in the transcription level of these five genes, which were consistent with their changes shown in 2-DE. These results showed that the disruption of the irpT gene may increase the nisin resistance level by regulation of these five genes expression.The dTDP-L-rhamnose synthase is involved in the synthesis of dTDP-L-rhamnose. which is an immediate precursor for cell wall polysaccharides backbone production. The constitution of the cell wall is very important in the development of nisin resistance in bacteria. High-performance liquid chromatography determination showed that the rhamnose content of cell wall polysaccharides was increased by 13.7% in L. lactis L31 compared with its parental strain. Moreover, overexpression of dTDP-L-rhamnose synthase in nisin-sensitive L. lactis MG1363 improved the nisin resistance. These results implied that the disruption of the irpT gene may increase the nisin resistance level partially by up-regulation of RmlD protein expression.Through the in-depth study of nisin resistance related genes, we can further explain the characterization of the network control mechanism of nisin resistance. These results may be useful for constructing strains with increased production of nisin as the nisin resistance level is one factor restricting the concentration of produced nisin.
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