Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro andin vivo

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100 -23, was determined, on the basis of nucleotide and deduced protein sequenc e data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located o n a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin- selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poo rly maintained (<33% resistant cells) in the L. reuteri population inhabiti ng the murine gastrointestinal tract, Stable maintenance (>90% resistant ce lls) of pGT232-derived plasmids in the lactobacillus population in vivo req uired an additional 1.0-kb sequence which contained a putative single-stran d replication origin (SSO), The SSO of pGT232 is believed to be novel and f unctions in an orientation-specific manner.