Role of -35 sequence and its cooperativity with vir-box for the expression of virE gene

To elucidate the role of the -35 sequence and its cooperativity with vir box in the expression of the virE gene, various mutants were constructed by either site-directed mutation or deletional mutation of the virE promoter. The expression level of pHBAV, a mutant where its putative -35 sequences (CCGAGT) have been substituted with the consensus -35 sequences of the Escherichia coli promoter (TTGACA), was increased by 386%. pECHV, containing the conserved -35 sequence but lacking the vir box and the 5'-half of the imperfect dyad symmetry region (DSR) showed an increase of 286% in its promoter activity. pESHV, containing the conserved -35 sequence but lacking the complete 5'-upstream region from the mid-region of imperfect DSR, exhibited 244% of the native virE promoter activity. pHBCA, containing the conserved -35 sequence but destroying the vir box, was constructed by substitution of A, C, T at the positions -62, -63, and -65 on the vir-box to T, A, C, respectively. These mutations increased promoter activities by 319%. On the other hand, when the vir box was mutated from imperfect DSR to almost perfect DSR with T to A and G to T substitutions at -60 and -61 positions of the virE promoter containing the conserved -35 sequence (pHBNA), a higher activity of 671% was observed. These results demonstrate that when the putative -35 sequence of virE promoter is replaced with the consensus -35 sequence, the virE gene can be expressed independently without the binding of VirG protein to the vir-box and/or the induction of acetosyringone. Moreover, the presence of an almost perfect dyad symmetry of the vir-box can increase the expression of virE synergistically with the consensus -35 sequence.