Differences in Induction of Ti Plasmid Virulence GenesvirGandvirD, and Continued Control ofvirDExpression by Four External Factors

Abstract

We investigated external factors controlling the expression of virG and virD in the vir region of Ti plasmids. virG expression was measured by immunological demonstration of the protein in western blots. Cells grown in YEB medium at pH 7.2 contain little if any of the protein. Induction requires two factors, temperatures below 28.degree.C and sucrose; the protein is detectable after less than 1 hr. Expression in the presence of these factors is stimulated by acetosyringone and acidic pH. The regulation of virD was investigated with a virD promoter-reporter (galK) gene construct in the presence of a Ti plasmid containing the complete vir region (pGV2260). Temperatures below 28.degree.C, sucrose, acetosyringone, and acidic pH are strict requirements for induction, indicating that the expression of virD is controlled more stringently than is virG. Preincubation of the cells with combinations of three of these four factors did not significantly reduce the length of the virD lag phase, indicating that all four factors must be present simultaneiously for the initiation of expression. Removal of any one of the four factors after the onset of induction stopped or reduced further expression. This indicates that the activation of the vir genes does not represent an irreversible commitment to induction.