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Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin
1Department of Biotechnology, University of New South Wales, Sydney, Australia.
Abstract:
A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.
Insights
A novel 60 kb conjugative plasmid, pND300, in Lactococcus lactis confers nisin resistance and mobilizes other plasmids. Its nisin resistance genes are similar to those in nisin production transposons.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Nisin resistance in Lactococcus lactis is crucial for industrial applications.
- Conjugative plasmids play a significant role in bacterial adaptation and gene transfer.
Purpose of the Study:
- To characterize the 60 kb conjugative plasmid pND300 from Lactococcus lactis M189.
- To elucidate the genetic basis of nisin resistance encoded by pND300.
- To investigate the mobilization capabilities of pND300.
Main Methods:
- Plasmid isolation and characterization (60 kb).
- Subcloning of nisin resistance determinant (12 kb to 10.4 kb).
- Molecular analyses including restriction analysis, PCR, Southern hybridization, and DNA sequencing.
Main Results:
- pND300 confers nisin resistance and exhibits conjugative transfer capabilities, mobilizing lactose utilization plasmids.
- The nisin resistance determinant is approximately 10.4 kb and shares similarity with known nisin production transposons.
- pND300 encodes key nisin resistance genes (nisR, nisK, nisF, nisE, nisG) and is flanked by IS946 insertion sequences.
- A putative promoter controlling nis gene expression was identified upstream of nisR.
Conclusions:
- pND300 is a novel conjugative plasmid in Lactococcus lactis encoding nisin resistance.
- The genetic organization of nisin resistance genes on pND300 provides insights into nisin resistance mechanisms.
- IS946 elements likely play a role in the structure and regulation of the nis genes on pND300.