Transfer RNA modification, temperature and DNA superhelicity have a commontarget in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene

Full expression of the virulence genes of Shigella flexneri requires the pr esence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2-methylthio-N-6-isopentenyladenosine (ms(2)i(6)A37, adjacent to and 3' of the anticodon)]. The synthesis of the se two nucleosides depends on the products of the tgt and miaA genes respec tively. We have shown that the intracellular concentration of the virulence -related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of Vi rF in the tgt and the miaA mutants suppressed the less virulent (tgt) or th e avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulenc e phenotypes at 30 degrees C, but forced synthesis of VirF at 30 degrees C induced the virulence phenotype at this low temperature. In addition, remov al of the known gene silencer H-NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a viru lent phenotype at 30 degrees C. Conversely, decreased expression of VirF at 37 degrees C induced by the addition of novobiocin, a known inhibitor of g yrase, led to an avirulent phenotype. We conclude that tRNA modification, t emperature and superhelicity have the same target - the expression of VirF - to influence the expression of the central regulatory gene virB and there by the virulence of Shigella. These results further strengthen the suggesti on that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacte rial translational machinery in the regulation of the expression of virulen ce genes which appears here quantitatively as important as the well-establi shed regulation on the transcriptional level.