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

Abstract

Aims: This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production. Methods and Results: The highest transformation efficiency, 1·1 × 102 transformants μg−1 of input transposon DNA, was achieved when cells were grown to an OD600 of 0·5 in the presence of 1·5% of glycine and treated with 20 μg 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 ∼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. Conclusions: 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. Significance and Impact of the Study: Expanding the Mu transposition‐based mutagenesis to Lactococci adds a new tool for studies of industrially important bacteria.

Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes

“…The yield is similar to the yields obtained with Streptococcus suis (100 CFU/g DNA) and Lactococcus lactis (110 CFU/g DNA), lower than the yields obtained with Staphylococcus aureus (20,000 or 12,000 CFU/g transposon DNA depending on the strain), and higher than the yield obtained with Streptococcus pyogenes (10 CFU/g transposon) (26,46). Compared to the efficiency in gram-negative bacteria, the efficiency in C. perfringens is 2 to 3 orders of magnitude lower (21).…”

“…Although a variety of transposon mutagenesis methods are available for gram-positive bacteria (4,37,41,43), the inherent species nonspecificity, as well as the lack of mobility of the integrated transposon, makes the bacteriophage Mu-based transposon delivery system a system of choice for a variety of species (16,26,46). The Mu transposition approach includes in vitro assembly of a complex between the transposon DNA and the transposase enzyme, the transpososome, followed by delivery of the transpososome into the recipient cells.…”

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

“…Up today, in vitro-assembled Mu transpososome complexes have been efficiently applied for the in vivo random insertion of recombinant DNAs into different bacterial genomes by the “nick–join–repair” pathway (Haapa et al 1999; Laasik et al 2005; Lamberg et al 2002; Lanckriet et al 2009; Pajunen et al 2005; Savilahti et al 1995; Savilahti and Mizuuchi 1996; Tu Quoc et al 2007; Wei et al 2010; Wu et al 2009). Moreover, efficient Mu transpososome-based integration has been verified even in yeast, mouse, and human genomes (Paatero et al 2008; Turakainen et al 2009).…”

Nisin-selectable food-grade secretion vector for Lactococcus lactis

“…PCR products were purified with the EZNA cycle-pure kit (Omega Bio-tek) and sequenced (Institute of Biotechnology, Helsinki, Finland). Plasmids were isolated with the plasmid mini kit (Omega Bio-tek) with following modifications: lysozyme was used at 1 mg/ml in solution I followed by incubation at 37°C for 1 h. Electro transformation was performed as described previously (Wu et al 2009). …”