g., an initiator protein) were provided in trans. To more buy GSK2126458 precisely locate the oriV within repC, we cloned a collection of internal segments of repC into the suicide

vector pDOP (Figure 1). This collection was conjugated into an R. etli strain containing the parental plasmid (CFNX101) as the source of all the trans elements required for replication, but we were unable to obtain transconjugants. To determine if the activation of oriV requires transcription (i.e., the repC mRNA also acts as a replication primer), we constructed a pDOP derivative that contained a repC gene but lacked a SD sequence (pDOP-Cs/SD) (Figure 1). This plasmid was also incapable of replicating in R. etli CFNX101. Similarly, the two plasmids with repC frame-shift mutations, pDOP-CBglII and pDOP-CSphI, were also conjugated into R. etli CFNX101 without success. Overall, these results indicate that RepC exerts its action in cis. RepC as an incompatibility factor Plasmid incompatibility, or the inability of two replicons to coexist in the same cell line, results from the sharing of elements involved in plasmid replication, partitioning or control [30]. The repC open reading frame of p42d, when cloned

in a vector capable of replicating in R. etli, CFNX101, can coexist with p42d [8]. However, all of our attempts to introduce the construct pDOP-C into R. etli CFNX101 www.selleckchem.com/products/idasanutlin-rg-7388.html failed. In contrast, CFNX101 transconjugants carrying a similar construct (pDOP-CsA) that contained the repC gene pSymA of S. meliloti 2011 were easily obtained. The frequencies with which CFNX101/pDOP-CsymA and CFNX107/pDOP-CsymA transconjugants were obtained were similar (average 5 × 10-3). Moreover, the plasmid profiles of the transconjugants showed that pDOP-CsA replicated in these strains as an independent entity. These observations indicate that pDOP-C and

its parental plasmid p42d are incompatible, while that of pDOP-CSymA and p42d are compatible. The RepC protein of S. meliloti 2011 Dynein pSymA shares 54% identity with the p42d RepC protein, and both proteins have very similar secondary structures (Figure 6). To map the RepC regions of p42d that are involved in plasmid incompatibility, a collection of hybrid genes containing fragments of the repC genes from S. meliloti pSymA and R. etli p42d were constructed. A schematic representation of the hybrid genes and their properties is shown in Figure 7. The hybrid genes were designed so that none of the predicted alpha-helix and beta regions of the repC products were disturbed. The hybrid genes were cloned into pDOP under the Plac promoter and transferred by conjugation into R. etli CFNX107 to determine their ability to replicate autonomously and into R. etli CFNX101 to test if they were able to replicate without the interference of p42d.