Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-by plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus-Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the Slayer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein. (C) 2002 Elsevier Science (USA). All rights reserved.