Abstract
We previously described a plasmid of Agrobacterium spp., pAoF64/95, in which the quorum‐sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum‐sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other repABC plasmids. However, traR, unlike its counterpart on Ti plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP, that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four‐gene mocC operon as well as tmsP, which is the distal gene of the eight‐gene motA operon. As judged by a bacterial two‐hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM‐binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon.
Highlights
Many members of the family Rhizobiales harbor very large extrachromosomal elements
We demonstrate that transcription of both traR and traM is constitutive and does not respond to growth with the conjugative opine
The strain that harbored pSRKGm::mrtR demonstrated IPTG-dependent repression of β-glucoronidase activity, even when the lowest amount of IPTG was added (Figure 5c, and data not shown). These results indicate that MrtR represses the mocC promoter and mannopine catabolism
Summary
Many members of the family Rhizobiales harbor very large extrachromosomal elements. Some of these plasmids, which replicate using a conserved repABC system, confer on their host bacteria defining biological functions. This regulator loses affinity for its operator sequences in the acc and arc promoter regions when it binds arabinose-2-phosphate, the first intermediate in catabolism of the two agrocinopine opines (El Sahili et al, 2015) In both Ti plasmids, the two signals form a regulatory cascade with the conjugative opine controlling expression of the quorum-sensing system which, in turn, is responsible for inducing transcription of the tra regulon (Figure S1). We provide data that support a model in which the small TraR-like protein, which we name TmsP, interacts with TraM thereby freeing up sufficient amounts of functional TraR to activate transcription of the tra regulon
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