Mu transposition complex mutagenesis inLactococcus lactis- identification of genes affecting nisin production

Abstract

This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production. The highest transformation efficiency, 1.1 x 10(2) transformants microg(-1) of input transposon DNA, was achieved when cells were grown to an OD(600) of 0.5 in the presence of 1.5% of glycine and treated with 20 microg ml(-1) ampicillin for 60 min. Three insertions affecting nisin production, which were identified at nisB, fhuR, and rpiA genes, were screened from a library of approximately 2000 erythromycin-resistant transformants using a nisin bioassay method. NisB is part of the nisin biosynthetic machinery, explaining the loss of nisin production in nisB mutant. FhuR is a transcription regulator involved in sulphur acquisition. Inactivation of fhuR presumably results in a low cellular cystein level, which affects nisin biosynthesis that involves utilization of cystein. RpiA is involved in pentose phosphate pathway and carbon fixation. The rpiA mutant showed reduction in nisin production and slow growth rate. The results showed that Mu transposition complex mutagenesis can be used to identify genes in L. lactis. Three genes involved in nisin production were identified. Expanding the Mu transposition-based mutagenesis to Lactococci adds a new tool for studies of industrially important bacteria.