The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-h omoserine lactone, We isolated four Tn5-induced mutants of A. tumefaciens C 58 deficient in Trap-mediated activation of tra genes on pTiC58 Delta accR. These mutations also affected the growth of the bacterium but had no detec table influence on the expression of two tester gene systems that are not r egulated by quorum sensing. In all four mutants Tn5 was inserted in a chrom osomal open reading frame (ORF) coding for a product showing high similarit y to RNase D, coded for by md of Escherichia coil, an RNase known to be inv olved in tRNA processing. The wild-type allele of the md homolog cloned fro m C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens md, but none of these genes exert ed a detectable effect on the expression of the tra reporter, In the mutant , traR was expressed from the Ti plasmid at a level about twofold lower tha n that in NT1. The expression of tra, but not the growth rate, was partiall y restored by increasing the copy number of traR or by disrupting traM, a T i plasmid gene coding for an antiactivator specific for TraR, The mutation in md also slightly reduced expression of two tested vir genes but had no d etectable effect on tumor induction by this mutant. Our data suggest that t he defect in tra gene induction in the mutants results from lowered levels of TraR, In turn, production of sufficient amounts of TraR apparently is se nsitive to a cellular function requiring RNase D.