Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

Abstract

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed LactobaciUus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or BaciUlus subilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIll, EcoRI, XhoH, and Hpall, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.

Genetics of bacteriocins produced by lactic acid bacteria

“…Nis' has been found on various plasmids in lactococci and is commonly associated with other traits (such as phage-defense mechanisms, lactose-fermenting ability) that are subject to conjugal mobilization [116][117][118][119]. Genetic characterization of the nsr gene from pNP40 identified an ORF of 318 codons that could encode a protein of 35 035 Da [114,116].…”

“…The nsr gene was the first selection marker used for development of food-grade vectors in LAB [114,116,119,121,122]. yon Wright et al [119] first employed a Nis r determinant in a food-grade vector designed entirely from lactococcal DNA sequences, pVS40 (7.8 kb) contained a lactococcal origin of replication and a 3.8-kb EcoRI fragment cloned from a 46-kb Nis' plasmid found in L. lactis subsp, lactis 1(I.084. Froseth et al [116] initially cloned the Nis r determinant and used it as a secondary marker after erythromycin-resistant (Era r) transformants were selected.…”

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…Nisin has been licensed in a number of countries around the world because of its antimicrobial activity against a broad range of Gram-positive bacteria, including Staphylococcus aureus and Listeria species. The first food-grade plasmids based on bacteriocin resistance contained the nisin resistance gene [90,91]. Similarly, selection of L. lactis transformants was obtained with a plasmid containing the nisin immunity gene nisI as a selection marker [92].…”

Food-grade Selection Markers in Lactic Acid Bacteria

“…The nisin resistance of Lactococcus lactis always base on two factors: (i) for nisin-producing Lactococcus lactis strains, NisI seems to play an important role in nisin immunity; (ii) for unnisin-producing Lactococcus lactis strains, they encode the nisin resistance determinant (nisr) (14). The first food-grade systems based on bacteriocin resistance were plasmids pVS40, pFM011, and pFK012 containing the nisin resistance marker nisr from Lactococcus lactis (9,11,12). Electroporating the plasmids with nisr gene into hosts could make the hosts grow in medium added a certain content of nisin.…”