Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: Construction of an Escherichia coli– Lactobacillus shuttle vector

Abstract

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a −10 region, and a −35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.