Characterization of endogenous plasmids from Lactobacillus salivarius UCC118

Department of Microbiology, Alimentary Pharmabiotic Centre, University College Cork, Ireland

^* To whom correspondence should be addressed. Email: pwotoole{at}ucc.ie.

	Abstract

The genome of Lactobacillus salivarius UCC118 comprises a 1.83 Mb chromosome, a 242 kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a TA (toxin-antitoxin) system composed of pemI/pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector-derivatives of pSF118-20 were generated that included the replication region (pLS203), and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in L. plantarum, L. fermentum, and in the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and Green Fluorescent Protein (GFP), and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.