Characterization of a Cryptic Plasmid pD403 from Lactobacillus plantarum and Construction of Shuttle Vectors Based on its Replicon

A cryptic plasmid pD403 was isolated from Lactobacillus plantarum D403 derived from fermented dairy products. It was 2,791 bp in size with a G+C content of 37%. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. ORF1 (318 amino acids) was identified as a replication protein (RepA). ORF2 (137 amino acids) shared 31% similarity with the transcriptional regulator of Ralstonia pickettii 12D. Functional investigation indicated that ORF2 (Tra) had the ability of improving the transformation efficiency. The origin of replication was predicted, suggesting that pD403 was a rolling-circle-replication (RCR) plasmid. An Escherichia coli/Lactobacillus shuttle vector pCD4032 was constructed based on the pD403 replicon, and proved to be successfully transformed into various lactobacilli including Lactobacillus casei, Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus brevis. The transformation efficiencies were ranged from 1.3 × 10² to 7 × 10⁴ transformants per microgram DNA. Furthermore, an expression vector pCD4033 was developed with the promoter of the lactate dehydrogenase from Lactobacillus delbrueckii 11842. The green fluorescent protein (gfp) as a reporter was expressed successfully in various lactobacilli tested, suggesting that the expression vector pCD4033 had the potential to be used as a molecular tool for heterologous gene cloning and expression in lactobacilli.