Ability of IncP‐9 plasmid pM3 to replicate in Escherichia coli is dependent on both rep and par functions

Abstract

Ability of IncP-9 plasmid pM3 to replicate in SummaryIncP-9 plasmids are common in Pseudomonas species and can be transferred to other Gram-negative eubacteria but tend not to be stably maintained outside their natural host genus. A 1.3 kb ori V-rep fragment from IncP-9 plasmid pM3 was sufficient for autonomous replication in Pseudomonas putida but not in Escherichia coli . Replication of ori V-rep in E. coli was restored when additional rep was provided in trans , suggesting that the replication defect resulted from insufficient rep expression from its natural promoter. A promoter deficiency in E. coli was confirmed by reporter gene assays, transcriptional start point mapping and mutation of the promoter recognition elements. Dissection of the pM3 mini-replicon, pMT2, showed that this replication deficiency in E. coli is suppressed by additional determinants from its par operon: ParB, which can be supplied in trans , and its target, the par operon promoter, required in cis to ori V-rep . We propose that ParB binding to its target either changes plasmid DNA and thus promoter conformation or by spreading or looping contacts RNAP at the rep promoter so that rep expression is sufficient to activate ori V.

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in <i>Pseudomonas</i> within Natural Environments and Microcosms

“…IncP-9 plasmids also have this system (parABkorAparC is located on oriV-rep upstream), although detailed functional analyses have not been reported. Intriguingly, the oriV-rep system of IncP-9 cannot function in E. coli unless it is accompanied by at least part of the par region in cis (88). As for the IncP-7 plasmids, products of parWAB are essential for their maintenance, although the parS site has not been determined; and the function of ParW is still unclear (92).…”

“…IncP-9 plasmids have long been known to replicate in a wide range of bacteria of the genus Pseudomonas, but many are unstable and replication-defective at higher temperatures in other taxa such as E. coli (10). The oriV-rep fragment (oriV is located on rep upstream) of minimal replicon of the IncP-9 plasmid pMT3 is sufficient for replication in P. putida but not E. coli because of a difference in rep promoter activity between these two hosts (88). The amount of Rep protein is suggested to be important for the replication of IncP-9 plasmids (88).…”

Predicting Plasmid Promiscuity Based on Genomic Signature

“…The replication initiation proteins used to retrieve the plasmids and their corresponding reference plasmids were RepA from the IncA/C plasmid pRA1 (34,45), RepA from the IncL/M plasmid pCTX-M3 (52), Rep from the IncP-9 plasmid NAH7 and pWW0 (60,61), RepC from the IncQ plasmid RSF1010 (50,59), and RepB from the IncU plasmid pRA3 (41). The PromA (74) and Ri/Ti (68) plasmids used here were retrieved from the literature and include those representatives whose genome sequences were available in GenBank in July 2009.…”

The Centromere Site of the Segregation Cassette of Broad-Host-Range Plasmid RA3 Is Located at the Border of the Maintenance and Conjugative Transfer Modules

“…The presence of similar cassettes carrying the regulatory korA gene nearby the partition module was also demonstrated in plasmids of the IncP-9 group (14,46) and in environmental plasmids recently defined as the PromA group, e.g., pIPO2 (50), pSB102 (45), pMOL98 (54), and pTer331 (36). This study shows that the segregation region of RA3 has a number of structural and regulatory properties that clearly distinguishes it from the equivalent regions of RK2 and R751 that have been analyzed most thoroughly.…”