To characterize the transposition mechanism of the IS-element IS10R and study how this element is involved in gene disruption in Lactococcus lactis.Methods and Results
The gene flciA confers immunity against lactococcin A in lactococci. However, the immunity function was lost when flciA was co-expressed with the regulator gene nisR on a plasmid in L. lactis NZ9000. By PCR and DNA sequencing, it was revealed that flciA in immune-negative transformants was disrupted by the IS-element IS10R. Such gene disruption did not occur when flciA was expressed alone nor when the plasmid-located nisR was mutated, suggesting that nisR is directly involved in the transposition. The sequence 5′-CACTTAACC-3′, which was found in flciA and at both ends of the inserted IS10R, was identified as target site by site-directed mutagenesis.Conclusions
IS10R transposes in L. lactis NZ9000 in a nisR-dependent fashion and employs the sequence 5′-CACTTAACC-3′ as integration site.Significance and Impact of the Study
To our knowledge, this is the first time IS10R and aspects of its transposition are described in the industrial important bacterium L. lactis. The highly controllable insertion of IS10R into a target site might present a great potential as a gene disruption system.