Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

Abstract

An 8.8-kb plasmid (pND302) was identified in Lactococcus lacti spp lactis M71 which encodes cadmium resistance (CdR). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, CdR should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (NisR) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing NisR and CdR, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10(3)/micrograms DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci.

Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker

“…Antibiotic resistance has been rejected as a selectable marker for microorganisms destined for use in food production because of the risk of promoting antibiotic resistance in human intestinal microflora. Hence, the identification of alternative selectable markers (e.g., bacteriocin immunity and heavy metal resistance) that can be considered safe for use in food has received considerable interest recently (7,18,23,36,47,50,51).…”

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2.

“…Recently, we reported the construction of cloning vectors based on a lactococcal plasmid (pND302) encoding cadmium resistance (Liu et al, 1996). The FG2 plasmid replicon was found to be compatible with that of pND3o2 (data not shown).…”

“…Plasmid isolation from L. lactis was as described by Anderson and McKay (1983). Electroporation of L. lactis was performed as described previously (Liu et al, 1996) using a Bio-Rad Gene Pulser apparatus (CA, U.S.A.). Other techniques used were essentially as described by Sambrook et al (1989).…”

Insertional Inactivation of Determinants for Mg 2+ and Co 2+ Transport as a Tool for Screening Recombinant Lactococcus Species Clones

“…Examples include resistance to the heavy metal cadmium (10) and resistance to the bacteriocin nisin (20), as well as several complementation markers that include the amber suppressor supD (18), the lacF gene, involved in lactose metabolism (11), or the thymidylate synthase gene, thyA (16,19). However, the presence of inherent background resistances associated with many of these food-grade markers has placed limitations on their use.…”