Development of surface adhesion in Caulobacter crescentus

Abstract

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.

FIG. 1.

Binding assays comparing cell attachment to and detachment from glass without growth and energy. (A) A binding assay was used to compare the effect of chemicals and temperature on the attachment of C. crescentus to glass coverslips. Cells were incubated under the following conditions: (a) no chemicals added; (b) chloramphenicol, 2 μg/ml; (c) sodium azide, 0.05% (wt/vol); (d) formaldehyde, 1.7% (wt/vol); (e) no chemicals added, 4°C. (B) A binding assay was used to determine the effect of 0.05% sodium azide and 1.7% formaldehyde on cells already attached to glass coverslips. Glass coverslips were exposed to exponentially growing cell cultures for 5.5 h starting at an OD₆₀₀ of 0.15. At this time sodium azide and formaldehyde were added, and incubation was continued for the indicated time. (a) No chemicals; overnight incubation. (b) No chemicals and incubation for 5.5 h. (c) Sodium azide at 0.05% (wt/vol) and overnight incubation. (d) Formaldehyde at 1.7% (wt/vol) and overnight incubation.

FIG. 2.

Quantification of holdfast flagella and pilus mutants attached to polystyrene. Exponentially growing cells were allowed to attach to polystyrene wells, and the wells were washed and stained with crystal violet. The bound crystal violet was solubilized with methanol, and color intensity was measured at 589 nm.

FIG. 3.

Microscopic analysis comparing attachment patterns of wild-type cells and flagellar and pilus mutants over the course of 4.5 h. Overnight cell cultures were diluted to an OD₆₀₀ of 0.15 and placed in the wells of a tissue culture dish containing plastic coverslips. Incubation proceeded at 30°C for 4.5 h with shaking. Beginning at 1.5 h, plastic coverslips were examined at 1-h intervals with a Nikon Eclipse E800 microscope (40×).

FIG. 4.

Surface attachment throughout the cell cycle of a synchronized population of C. crescentus. (A) The crystal violet assay of bound cells is represented by circles (averages of two assays presented as a percentage of the maximum cell attachment). A phage absorbance assay measuring the binding of phage φCbK was used to measure the presence of pili throughout the cell cycle (squares). (B) Immunoblot of flagellins throughout the cell cycle. (C) Diagram of the cell cycle as observed microscopically throughout the synchrony.