Characterization and sequence analysis of a small cryptic plasmid from Lactobacillus curvatus LTH683 and its use for construction of new Lactobacillus cloning vectors.

Lactobacillus curvatus LTH683, a strain originally isolated from raw sausage, contains the single cryptic plasmid called pLC2. The sequence and genetic organization of the complete 2489-bp plasmid pLC2 was determined and used as the basis for construction of a series of vectors useful in Lactobacillus strains. The major parts of pLC2 nucleotide sequence could be aligned with other plasmids from gram-positive bacteria replicating by a rolling circle mechanism of replication (RCR). Direct evidence for a RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. Three protein-coding sequences could be predicted and the corresponding proteins were detected after in vitro transcription/translation of pLC2 plasmid DNA. ORFs 1 and 3 showed minor homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and its corresponding target sequence, the plus origin, were similar to replication regions of other gram-positive bacteria plasmids like pLS1, pWV01, and pE194. Upstream of the ori+ site, in a noncoding region, which was nonessential for replication, strong homology to other Lactobacillus plasmids like pC30i1, pLP1, pLJ1, and pLAB1000 could be detected. A palindromic sequence predicted to be the minus origin of replication was localized there. Small vectors (3213 bp) suitable for cloning in lactobacilli were constructed based on a 1635-bp DNA fragment of pLC2, containing the region necessary for replication, marked with the chloramphenicol resistance gene and a multiple cloning site.