16] - Genetic Dissection of Signaling Through the Rcs Phosphorelay

Abstract

The Rcs phosphorelay, consisting of a hybrid sensor kinase, a phosphotransferase, and a response regulator, regulates a large number of bacterial functions. These include capsule production, the target originally defined for these regulators, a small regulatory RNA, and a growing list of additional genes, many of unknown function. At the core of this phosphorelay is the response regulator RcsB that activates the expression of the target genes. In addition to RcsB, some but not all of these targets require a co-regulator. One such co-regulator is RcsA, which has not been described as working except with RcsB; RcsA is itself regulated at both the transcriptional and post-transcriptional levels. Signaling to the system is also complex, and numerous plasmids, mutations, and environmental conditions have been described as activating this system. Activation of the system on cell surfaces and the nature of some of the regulated functions suggest a role for this phosphorelay in biofilm formation. Here, we describe reporters and mutants that allow the genetic dissection of the system from two directions. In cases where a condition activates the system, for instance, causing an increase in capsule synthesis (a phenotype easily observed in colonies), specific tests can identify at what stage the signal feeds into the system. In cases where a target of the phosphorelay is identified, specific tests can define the genetic requirements for regulation of the target. Finally, in cases where overproduction of capsule interferes with other studies, mutants allow the study of cells in the absence of capsule formation.