Abstract
Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulating V. cholerae biofilm dispersal to ameliorate disease.
Highlights
Regulator of biofilm formation, and that expression of vibrio polysaccharide biosynthetic genes are required [15,16,17]
V. cholerae cells are inoculated onto glass coverslips at low cell density, and brightfield timelapse microscopy is used to monitor biofilm progression
In other bacteria, straight-swimming mutants are deficient in traversing fluid-filled porous media compared with WT organisms that can reorient [36]. These results indicate that chemotaxis itself is not required for biofilm dispersal, but the chemotaxis machinery facilitates random reorientation events that allow V. cholerae cells to navigate a porous biofilm matrix
Summary
Regulator of biofilm formation, and that expression of vibrio polysaccharide (vps) biosynthetic genes are required [15,16,17]. The strategy of characterizing constitutive biofilm formers, while successful for uncovering factors that promote biofilm formation, has necessarily precluded studies of biofilm dispersal. We used a microscopy assay that allowed us to monitor the full wildtype (WT) V. cholerae biofilm lifecycle. We combined this assay with high-content imaging of randomly mutagenized WT V. cholerae to identify genes required for biofilm dispersal. Investigation of the proteins encoded by the genes allowed us to characterize the signaling relays, matrix-digestion enzymes, and motility components required for biofilm dispersal, a key stage in the lifecycle of the global pathogen V. cholerae
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