Cohesive properties of the caulobacter crescentus holdfast adhesin are regulated by a novel c-di-GMP effector protein

Many bacteria grow in aquatic environments such as oceans, lakes and rivers. These environments pose special challenges, as nutrient availability is poor and hard to exploit due to constant water flow. Therefore, most bacteria grow on surfaces and have developed mechanisms that anchor them on an adequate nutrient source in the moment of encounter. Yet, they need to effectively disperse when conditions become unfavorable. The switch between motile and sessile lifestyles is controlled by the global second messenger c-di-GMP, which suppresses motility and promotes sessility. Here, we use the aquatic bacterium Caulobacter crescentus as a model to dissect c-di-GMP mediated lifestyle changes and surface attachment. C. crescentus has a biphasic life cycle that comprises both a motile and a sessile phase. A motile swarmer cell can develop into a sessile surface attached stalked cell by expressing a polar exopolysaccharide-based structure called the holdfast that shows remarkably strong adhesive properties. Recent studies have shown that holdfast biogenesis requires c-di-GMP and is initiated either upon surface contact or as part of the developmental program. However, the underlying molecular mechanisms are not understood.
In an unbiased screen for c-di-GMP binding proteins in C. crescentus we identified a novel c-di-GMP effector of unknown function and confirmed specific binding to c-di-GMP in vitro. Orthologous genes of this effector were found in different phyla and showed a strong association with exopolysaccharide synthesis genes. Its importance in holdfast biogenesis could be experimentally confirmed and the protein was named HfsK following the terminology used for holdfast synthesis proteins. Cells deficient in HfsK produced holdfast but could not retain the adhesin on the cell envelope, resulting in a strong surface adhesion defect and failure in surface colonization. Furthermore, a strong deformation of the mutant holdfast was observed when it was subject to shear stress, indicating a decrease in holdfast cohesion forces. HfsK is a member of an uncharacterized subclass of Gcn5-related-N-acetyltransferases, a diverse enzyme family that could potentially acylate the holdfast exopolysaccharide. Mutations in genes encoding holdfast anchor proteins that are predicted to form membrane-anchored filaments exhibited a similar phenotype. Based on our findings we propose a model in which the anchor filaments and the holdfast form a strong interaction network that relies on HfsK-mediated chemical modification of the holdfast exopolysaccharide.
In accordance with its proposed function in holdfast biogenesis, HfsK predominately localized to the cell membrane. The protein delocalized into the cytosol for a short period during the cell cycle, coinciding with holdfast biogenesis and with an upshift of cellular c-di-GMP levels. HfsK mutants impaired in c-di-GMP binding remained membrane associated throughout the cell cycle indicating that c-di-GMP binding controls the localization of this protein. A role in HfsK control could be attributed to a short stretch of amino acids at the C-terminus. This part of the protein is involved in c-di-GMP binding and is required for HfsK localization and activity. Functional analysis revealed a clear correlation between HfsK activity and subcellular localization. HfsK mutants that remained membrane-associated were mostly found to be active, while variants exclusively localizing to the cytosol failed to support proper holdfast formation. We propose that c-di-GMP binding to the C-terminus of HfsK leads to its delocalization and concomitant inactivation.
Finally, we show that overexpression of the glycosyltransferase HfsJ restored holdfast biogenesis in a strain lacking c-di-GMP. This exposed HfsJ as catalyst of the rate-limiting step of holdfast biogenesis when c-di-GMP levels are low. Together with recent findings that HfsJ directly binds c-di-GMP this suggested furthermore that HfsJ is the main effector through which holdfast synthesis is activated upon a cellular upshift of c-di-GMP.
This work establishes two pathways through which c-di-GMP can activate and possibly modulate the holdfast adhesin of C. crescentus and provides novel insight into the basis of the holdfast strong adhesive properties.