Genetic Analysis ofVibrio choleraeMonolayer Formation Reveals a Key Role for ΔΨ in the Transition to Permanent Attachment

Abstract

A bacterial monolayer biofilm is a collection of cells attached to a surface but not to each other. Monolayer formation is initiated when a bacterial cell forms a transient attachment to a surface. While some transient attachments are broken, others transition into the permanent attachments that define a monolayer biofilm. In this work, we describe the results of a large-scale, microscopy-based genetic screen for Vibrio cholerae mutants that are defective in formation of a monolayer biofilm. This screen identified mutations that alter both transient and permanent attachment. Transient attachment was somewhat slower in the absence of flagellar motility. However, flagellar mutants eventually formed a robust monolayer. In contrast, in the absence of the flagellar motor, monolayer formation was severely impaired. A number of proteins that modulate the V. cholerae ion motive force were also found to affect the transition from transient to permanent attachment. Using chemicals that dissipate various components of the ion motive force, we discovered that dissipation of the membrane potential (⌬⌿) completely blocks the transition from transient to permanent attachment. We propose that as a bacterium approaches a surface, the interaction of the flagellum with the surface leads to transient hyperpolarization of the bacterial cell membrane. This, in turn, initiates the transition to permanent attachment.

Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms

“…In contrast, a flagellar motor mutant is completely defective in formation of both monolayer and multilayer biofilms. Furthermore, in mutants lacking both the flagellum and the flagellar motor, the phenotype of the flagellar motor mutant is dominant (187,328). This suggests that the flagellar motor plays a role in biofilm formation that is independent of that played either by flagellar motility or by the flagellar rotor.…”

“…We hypothesize that the bias toward permanent attachment is modulated by environmental signals, but to date, no such environmental signal has been elucidated. Recent evidence suggests, however, that changes in the membrane potential (⌬⌿) may alter the bias toward permanent attachment (328). In the sections below, we will outline what is known about the adhesive structures that mediate transient and permanent surface attachment, the transition to permanent attachment, and the monolayer transcriptome.…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…Evidence for this was provided by several research laboratories (92,155,156,163) and is exemplified by findings of Van Dellen et al (160) demonstrating that while V. cholerae O139 flagellar mutants show reduced attachment, they eventually form a robust monolayer biofilm. In contrast, monolayer formation was severely impaired in the absence of the flagellar motor.…”

“…The answer is a clear "maybe," as some genetic determinants appear to play only a temporary role. For example, loss of flagella has been shown in numerous bacteria to significantly reduce attachment, while continued incubation led to the formation of a robust monolayer and biofilms (160). Although P. aeruginosa ⌬retS mutants exhibit a hyperattachment phenotype, they are impaired in biofilm development upon continued incubation under flowing conditions.…”

Vibrio choleraePhosphoenolpyruvate Phosphotransferase System Control of Carbohydrate Transport, Biofilm Formation, and Colonization of the Germfree Mouse Intestine

“…Plasmids used for rescue experiments, which are listed in Table 1, were constructed as described previously (12,37), using the primers listed in Table 2. Briefly, either the native sequence or a truncated version of the targeted gene, excluding the start and stop codons, was amplified by PCR.…”