The replicon of pSW800 from Pantoea stewartii

A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes - repA and repB, which encode a 36.5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem-loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem-loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5′-GCACGGG-3′) located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem-loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, Inclα and IncL/M groups also regulates pSW800 replication.