Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

Abstract

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A twocomponent food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus : Organization, Sequence, Transcription, and Activity of the gal Gene Products

“…These included galK-, galE-, galM-, and lacS-specific probes. Prior to radiolabeling, probes were purified as described previously (6).…”

Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas

“…The finding that inactivation of pE by a single 4 bp insertion prevented replication clearly indicates that the polypeptide, rather than the nucleotide sequence, is required. The fact that the pE-disrupted plasmids failed to replicate in S. citri GII3 (a strain that produces PE from endogenous plasmids) suggests that PE, unlike other replication initiator proteins (Emond et al, 2001) might be a cis-acting protein, unable to support replication when provided in trans. In this experiment, however, the hypotheses of a retro-control system specific for S. citri GII3, or incompatibility between plasmids having identical replication regions, cannot be excluded (Bouet et al, 2007).…”

Characterization of Genes Involved in the Metabolism of α-Galactosides by Lactococcus raffinolactis

“…Transformations of E. coli (25) and L. lactis (16) were performed as described elsewhere. Plasmid DNA from E. coli and L. lactis was isolated as previously described (5,11). Total lactococcal DNA was obtained as described by Boucher et al (5).…”