A genome-wide analysis of adhesion inCaulobacter crescentusidentifies new regulatory and biosynthetic components for holdfast assembly

Abstract

Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is a carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We have leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multi-day time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This study defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces. Importance: Bacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast, which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.

The role ofCaulobactercell surface structures in colonization of the air-liquid interface

“…Flagella and pili are important factors for colonization of solid surfaces in C. crescentus (23,26) and other species (37)(38)(39). Recently published data provide support for complex interplay between the flagellum, type IV pili and control of holdfast development in C. crescentus (25,31,(40)(41)(42). Given the clear role of the pilus and flagellum in attachment to solid surfaces, and the regulatory connection between these structures and holdfast development, I tested the contribution of these appendages to C. crescentus pellicle development at the air-liquid interface.…”

“…∆flgH mutants, which lack a flagellum, colonize the air-liquid interface, albeit inefficiently and in a less uniform manner than wild type (Figures 5 and 8). It is known that the loss of flagellar genes including flgH and flgE results in a hyper-holdfast phenotype (31,42). In the context of pellicle development, the observed microcolonies of rosettes in the ∆flgH strain suggests that its hyper-holdfast phenotype can overcome the motility defect of this strain.…”

“…While the chemical composition of the holdfast material is not well understood, the genes required for its synthesis indicate it is a polysaccharide (29,30) that likely contains four distinct sugars (31).…”

“…Eventually, the surface of the culture medium became covered with a film that did not adhere efficiently to glass and fragmented into large pieces ( Figure 5, 72 hours). Though the non-motile ∆flgH strain was unable to actively move to the air-liquid interface, the hyper-holdfast phenotype of this strain (31,42) seemed to enable capture of cells that arrived at the surface by chance. This resulted in cell accumulation and formation of the observed microcolonies at this boundary.…”

“…It is notable that in a selection for mutants with surface attachment defects, these two mutants displayed distinct phenotypes; ∆pilA mutants had reduced surface attachment, while ∆cpaH mutants displayed enhanced surface attachment owing to increased holdfast synthesis (31).…”