Many bacteria can exist as surface‐attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small‐ or relatively large‐scale studies. Unlike assays involving continuous‐flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ⌬bifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ⌬bifA mutant but does not contribute to the observed swarming defect. The ⌬bifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.The gram-negative bacterium Pseudomonas aeruginosa is an important model organism for the study of bacterial surface interactions, including biofilm formation and surface-mediated twitching and swarming motilities. However, the precise molecular mechanisms required for transition from a planktonic mode of existence to that of a surface-associated lifestyle are only beginning to come to light.In the case of biofilm formation, microscopic studies, as well as genetic analyses, have shown that the initial surface attachment phase by P. aeruginosa proceeds in two distinct steps (8,20). In the first step, known as reversible attachment, cells are loosely attached via a single cell pole and may readily detach and return to the planktonic phase. In the second step, cells that are tethered by a pole become attached via the long axis of the cell body. Such cells, deemed irreversibly attached, are more firmly attached to the surface. Genetic studies of initial attachment have led to the identification of SadB, a key component required for this transition from reversible to irreversible attachment in P. aeruginosa (8).The ability to form a robust biofilm also requires the production of an exopolysaccharide (EPS) component of the biofilm matrix. Recent studies have identified genetic loci that are important for synthesis of an EPS component of biofilm matrix in several P. aeruginosa strains. In PA14, the pel genes are required for ...

Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ⌬sadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.

Many bacteria can exist as surface‐attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small‐ or relatively large‐scale studies. Unlike assays involving continuous‐flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms. Curr. Protoc. Microbiol. 22:1B.1.1‐1B.1.18. © 2011 by John Wiley & Sons, Inc.

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methylaccepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.Pseudomonas aeruginosa is an important model organism for the study of gram-negative biofilm development, yet little is known about the molecular mechanisms underlying the initial events leading to the surface interactions that characterize the early steps in bacterial biofilm formation. Microscopic observations (23,26,40,51) and genetic analyses (2) revealed two sequential events that lead to stable surface interactions. First, a bacterial cell pole contacts the surface in a process referred to as reversible attachment. This is a relatively unstable interaction, as reversibly attached bacteria can readily return to a planktonic existence. The second event is a transition from the polar association to one that is mediated by the long axis of the cell body, referred to as irreversible attachment. In P. aeruginosa, the only mutation known to block the transition from reversible to irreversible attachment is in the sadB gene (2).Another key aspect of biofilm formation by P. aeruginosa is the production of an extracellular matrix. In pseudomonads, this matrix is thought to be comprised of exopolysaccharides (EPS), DNA, and protein (19). The biofilm matrix has typically been credited with structuring the mature biofilm (4). Studies have identified the pel and psl loci as two sets of genes predicted to be involved in the production of the polysaccharide component of the matrix required for biofilm maturation by P. aeruginosa on abiotic surfaces, although only the pel gene cluster is found in P. aeruginosa strain PA14 (7,8,15,27), the focus of study in this report. Interestingly, recent studies suggest that the pel locus also plays a role in early biofilm formation. A pel mutant of P. aeruginosa PAK shows a strong attachment defect in a strain lacking type IV pili (48) and P. aeruginosa PAO1 with a mutation in the psl locus has a block in biofilm initiat...