Disrupting chemotaxis stimulates adhesion by recruiting a putative c-di-GMP effector to the Caulobacter crescentus cell pole

Abstract

Contact with solid surfaces activates signaling pathways that promote biofilm formation in many bacteria. The alphaproteobacterium Caulobacter crescentus uses its flagellum to sense surfaces and responds by synthesizing an adhesive called the holdfast. The C. crescentus surface sensing pathway can be activated by mutating genes required for the assembly of the flagellum or genes required for chemotaxis. However, flagellar assembly and chemotaxis mutations activate distinct surface sensing pathways that differ in the activation of the diguanylate cyclase PleD. Here, we used a genome-wide screen to identify cmrA (CCNA_02061) as a crucial determinant of hyperadhesion in the chemotaxis mutant ΔcheYII. Genetic analysis showed that cmrA is important for activation of PleD in a context-specific manner. It is dispensable in wild-type and late-stage flagellar (ΔflgH) mutant backgrounds but promotes adhesion in early-stage flagellar assembly (ΔfliF), chemotaxis (ΔcheYII) and stator (ΔmotB) mutant backgrounds. Fluorescently tagged CmrA displays a mostly cytoplasmic localization in genetic backgrounds where cmrA is dispensable for adhesion but localizes to the cell pole in backgrounds where it regulates adhesion. Structural modeling indicates that CmrA is a degenerate, catalytically inactive GGDEF/EAL domain containing protein, but cmrA alleles with mutated conserved c-di-GMP coordinating residues are unable to support hyperadhesion. Our results indicate that altering the directional switching of MotAB stators recruits CmrA to the cell pole where it activates PleD to drive surface adaptation. Ultimately, this work underscores the complexity of flagellar surface sensing by highlighting how the many rotational states of the motor stimulate distinct but overlapping c-di-GMP signaling pathways Importance Bacteria often transition from a free-swimming state to form surface-attached communities called biofilms. The flagellum allows bacteria to sense surface contact and activate biofilm formation, yet how distinct structural states of this complex machine trigger surface sensing remains poorly understood. In this study, we identify CmrA as a key signaling link that senses flagellar motor disruption and activates second-messenger signaling to promote cell adhesion in Caulobacter crescentus. Our findings demonstrate that bacterial surface sensing is not a simple binary switch. Instead, distinct mechanical perturbations to the flagellum engage specialized, overlapping signaling pathways to fine-tune surface adaptation. Understanding these nuanced pathways will inform strategies to manipulate biofilm formation for human benefit.