Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli

Abstract

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.

FIG. 1.

Physical map of pRV500 and sequence relationships with databases. The 13 ORFs of pRV500 are depicted by open arrows in the direction of their putative transcription. Direct repeats (iterons) located in the putative origin of replication oriA are shown. Segments having significant nucleotide sequence similarity or coding for products showing amino acid identity above 30% with sequences in databases are indicated.

FIG. 2.

Blocks of conserved residues between gene products of pRV500 and site-specific recombinases of the integrase family. Numbers at the left of the alignment indicate the coordinates of the first residue shown for each sequence. Residues identical or similar in at least half of the different sequences are shaded black or gray, respectively. See Table 1 for accession numbers for recombinases of Streptococcus pneumoniae TIGR4 and of Staphylococcus aureus MW2. Blocks indicated as patches and boxes are reported according to the results of Nunes-Düby et al. (42). The tetrad signature of the family is indicated by asterisks above the four relevant residues.

FIG.3.

(A) Structural organization of the ori region of pRV500. Coordinates of the schematized fragment are indicated at each end. The percentage of A-T composition of particular segments is indicated, and −35 and −10 boxes of the putative promoter of repA are represented. Each unit of identified direct (DR) or inverted (IR) repeats is depicted by an arrow. The sequence of the repeat units is indicated below. Lowercase letters give alternative sequence found in one of the units. Underlined letters indicate the nucleotides conserved in the iterons of pEF418 (AF408195) and pUCL287 (X75607). The motif TTGTCTGTTTAT, obtained by the juxtaposition of the repeated units, was also found to be present in the iterons of pMD5057 (AF440277), pLKS (AB035265), pRC18 (AF200347), and pLA105 (D49554) but not in those of pLA103 (D55703) or pSK639 (U40259). (B) Conservation of an N-terminal-to-central region in Rep proteins of three subfamilies of class A theta plasmids. Numbers at the left and at the right of the alignment indicate the coordinates of the first and last residues shown, respectively. The total number of residues for each protein is given in parentheses. Residue shading is as described in the legend for Fig. 2. Respective database accession numbers for pUCL287 to pPS10, in the order shown in the figure, are Q51863, P95741, Q47808, Q9S0J9, Q52180, Q07137, Q48681, P22308, and Q52546.

FIG. 4.

Sequence conservation among plasmids from LAB. Lfasta and Lalnview were used to detect and visualize nucleotide identity between plasmid sequences as schematized here (http://pbil.univ-lyon1.fr/lfasta.php). Gray shading and figures indicate sequence conservation and the percentage of nucleotide identity between two DNA segments. Segments with 100% nucleotide identity between pRV500 and pMD5057 or pRC18 are indicated by horizontal thick lines above the sequence lines. For easier visualization of sequence conservation, the DNA segment corresponding to orf13 of pRV500, which shares no similarity with other plasmids, was positioned underneath. Black and gray boxes represent stretches of direct repeats (DR) and palindromic sequences (IR), respectively. These palindromic sequences (about 40 bp) have higher conservation (>90% nucleotide identity) than that indicated according to the Lfasta program for the slightly larger region. orf genes putatively coding for recombinases and initiator proteins are indicated with dotted and hatched arrows, respectively, while those sharing amino acid similarity with orf6 of pRV500 are represented with arrows with vertical bars. Relevant portions of plasmids are schematized at the same scale here; for pLH1, pMD5057, and pLP9000, they are in the reverse direction relative to the entry in the database. Accession numbers for plasmid sequences (complete) were as follows: pLH1, AJ222725; pSRQ800, U35629; pUCL287, X75607; pMD5057, AF440277; pRC18, AF200347; pLP9000, AY096005.

FIG. 5.

pRV566, a shuttle vector able to replicate in E. coli and several lactobacilli. ORFs originating from pRV500 and pRV300 are represented in gray and black, respectively. R-11 and R-22 indicate the position of 11- and 22-bp directed repeats, respectively; ermAM and amp code for erythromycin and ampicillin resistance, respectively.

FIG. 6.

Hybridization pattern of plasmids extracted from lactobacillus strains poorly transformed or not transformed by pRV566. pRV566 was used as the probe. An equivalent amount (10 ng) of plasmid DNA extracted from various strains was run on the gel.