Genetic dissection of "Caulobacter crescentus" surface colonization

Abstract

During its biphasic life cycle Caulobacter crescentus switches from a planktonic to surface attached life style. This transition requires the continuous remodeling of the cell poles through the temporally and spatially coordinated assembly and disassembly of polar organelles like the flagellum, pili, and an adhesive holdfast. A genetic screen for mutants affected in surface binding and colonization led to the identification of various genes required for motility, pili, and holdfast biogenesis, suggesting a specific role for all three organelles in C. crescentus surface colonization. Several novel holdfast genes were identified, which are potentially involved in the synthesis and regulation of the polysaccharidic component of the holdfast. Quantitative surface binding studies during the C. crescentus cell cycle revealed that optimal attachment coincides with the presence of flagellum, pili, and holdfast at the same pole. This indicated that accurate temporal control of polar appendices is critical for surface colonization of C. crescentus and represents the first example for developmentally controlled bacterial surface adhesion. We have used genetic and biochemical analyzes to demonstrate that di-cyclic guanosine monophosphate (c-di-GMP) is a central regulatory compound involved in the timing of C. crescentus pole development. Mutants lacking the diguanylatecyclase PleD show a dramatic delay of holdfast formation during swarmer cell differentiation. In contrast, cells lacking the GGDEF-EAL composite protein CC0091 show premature holdfast formation, while overexpression of CC0091 also leads to a delayed appearance of holdfast. The observation that CC0091 is a c-di-GMP specific phosphodiesterase indicated that the antagonistic activities of PleD and CC0091 could be responsible for the correct timing of holdfast formation and flagellum ejection. Finally, our genetic screen identified a candidate for the c-di-GMP effector protein, which mediates holdfast synthesis in response to fluctuating levels of c-di-GMP. The glycosyltransferase CC0095 is strictly required for holdfast formation and its overexpression leads to premature holdfast synthesis. This and the observation that CC0095 is able to bind c-di-GMP lead to the hypothesis that holdfast synthesis is regulated via allosteric control of the CC0095 glycosyltransferase. These data provide the first example of a developmental process being regulated by the bacterial second messenger, c-di-GMP.