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 two-component 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.

FIG. 1

Functional analysis of the replication region of the lactococcal plasmid pCD4. A linear restriction map is shown at the bottom of the diagram. Putative ORFs (black arrows), cis-acting regions (light grey boxes), promoters (arrowheads), and terminators (stem-loops) shown on the restriction map were inferred from sequence analysis. Thick lines above the linear map represent the fragments cloned into pNC1. The names of recombinant plasmids containing these inserts are indicated on the left. The single break in pRL1 corresponds to the HindIII site used for the cloning of the complete pCD4, and the double breaks in pRL20 correspond to the deletion of ClaI fragments. Only those restriction sites used to generate pCD4 derivatives are indicated on the map. The replication phenotype (Rep) and the calculation of the stability of pCD4 derivatives in strain MG1363 (average percent plasmid loss per generation ± standard deviation) are indicated on the right. The vertical lines delimit the core replicon. ND, not determined.

FIG. 2

Sequence alignment of the oriT locus of lactococcal plasmids. Inverted and direct repeats are indicated above the sequence. IR1, IR2, and IR3 correspond to the inverted repeats that were identified on the functional transfer origins of pCI528 and pNZ4000 (36, 55). Inverted repeats IR4, IR5, and IR6 are present on the functional transfer origin of oriT1 and oriT2 (IR5 and IR6) defined on pNZ4000. The bold letters represent the core region of the R64 oriT locus. The NikA-binding site is boxed, and the nick site is indicated by the arrowhead.

FIG. 3

Sequence analysis of the minimal replicon of pCD4. (A) The restriction sites used to generate pCD4 derivatives are highlighted on the DNA sequence. The −35 and −10 boxes of repB promoter and RBSs are underlined. The reverse-complement sequence of the −35 and −10 boxes of the counter-transcript RNA promoter is double underlined. The AT-rich region is boxed, and the GC clusters are indicated with asterisks. Direct repeats within the AT-rich stretch are represented by double arrows, and the 22-bp iterated sequences repeated three and one-half times (iteron) are indicated by thick arrows above the sequence. Inverted repeats are represented by dashed arrows over the sequence. Protein translation of repB and of the truncated orfX is given below the DNA sequence. Amino acids in reverse video in the RepB sequence correspond to consensual residues with a plurality of at least 30 from an alignment of the amino acid sequence of 35 replication initiator proteins of lactococcal plasmids. The sequences used in the alignment were taken from GenBank. Amino acid motifs for the leucine zipper (positions 31 to 45), copy number control (129 to 144), and DNA binding (positions 215 to 241) are indicated with dashed underlines. The numbers on the left correspond to the nucleotide numbering of pCD4 as submitted to GenBank. The amino acid numbering of RepB is indicated on the right. (B) Helical wheel representation of the leucine motif of RepB. Hydrophobic residues are boxed, charged residues are indicated by a + sign, and the other residues correspond to uncharged polar residues.

FIG. 3

Sequence analysis of the minimal replicon of pCD4. (A) The restriction sites used to generate pCD4 derivatives are highlighted on the DNA sequence. The −35 and −10 boxes of repB promoter and RBSs are underlined. The reverse-complement sequence of the −35 and −10 boxes of the counter-transcript RNA promoter is double underlined. The AT-rich region is boxed, and the GC clusters are indicated with asterisks. Direct repeats within the AT-rich stretch are represented by double arrows, and the 22-bp iterated sequences repeated three and one-half times (iteron) are indicated by thick arrows above the sequence. Inverted repeats are represented by dashed arrows over the sequence. Protein translation of repB and of the truncated orfX is given below the DNA sequence. Amino acids in reverse video in the RepB sequence correspond to consensual residues with a plurality of at least 30 from an alignment of the amino acid sequence of 35 replication initiator proteins of lactococcal plasmids. The sequences used in the alignment were taken from GenBank. Amino acid motifs for the leucine zipper (positions 31 to 45), copy number control (129 to 144), and DNA binding (positions 215 to 241) are indicated with dashed underlines. The numbers on the left correspond to the nucleotide numbering of pCD4 as submitted to GenBank. The amino acid numbering of RepB is indicated on the right. (B) Helical wheel representation of the leucine motif of RepB. Hydrophobic residues are boxed, charged residues are indicated by a + sign, and the other residues correspond to uncharged polar residues.

FIG. 4

Circular maps of pVEC1 and pCOM1. Relevant features of the plasmids and restriction sites useful for cloning are indicated. Truncation in genes is indicated by apostrophes.

FIG. 5

Detection by hybridization of pVEC1 and pCOM1 in total DNA extracts from variants of L. lactis MG1363 harboring different combinations of plasmids by hybridization. (A) Agarose gel before Southern transfer. (B) Autoradiogram obtained after hybridization with a probe specific for pCOM1 prepared by labeling pNC1. (C) Autoradiogram obtained after hybridization with a probe specific for pVEC1 obtained by labeling a repB internal fragment of pSRQ900. Lanes: M, 1-kb DNA mass ladder (Gibco/BRL Life Technologies, Burlington, Ontario, Canada); 1, MG1363 total DNA/EcoRI; 2, MG1363(pCOM1, pVEC1)/EcoRI; 3, MG1363(pVEC1)/EcoRI; 4, MG1363(pCOM1, pVEC1)/ClaI; 5, MG1363(pVEC1)/NspV; 6, MG1363(pCOM1, pVEC1)/NcoI/XbaI; 7, MG1363(pVEC1)/NcoI. The molecular size of marker bands is indicated on the left in kilobases, and hybridization signals corresponding to pCOM1 and pVEC1 are indicated on the right.