- Top of page
- Results and discussion
- Experimental procedures
Biofilm development is conceived as a developmental process in which free swimming cells attach to a surface, first transiently and then permanently, as a single layer. This monolayer of immobilized cells gives rise to larger cell clusters that eventually develop into the biofilm, a three-dimensional structure consisting of large pillars of bacteria interspersed with water channels. Previous studies have shown that efficient development of the Vibrio cholerae biofilm requires a combination of pili, flagella and exopolysaccharide. Little is known, however, regarding the requirements for monolayer formation by wild-type V. cholerae. In this work, we have isolated the wild-type V. cholerae monolayer and demonstrated that the environmental signals, bacterial structures, and transcription profiles that induce and stabilize the monolayer state are unique. Cells in a monolayer are specialized to maintain their attachment to a surface. The surface itself activates mannose-sensitive haemagglutinin type IV pilus (MSHA)-mediated attachment, which is accompanied by repression of flagellar gene transcription. In contrast, cells in a biofilm are specialized to maintain intercellular contacts. Progression to this stage occurs when exopolysaccharide synthesis is induced by environmental monosaccharides. We propose a model for biofilm development in natural environments in which cells form a stable monolayer on a surface. As biotic surfaces are degraded with subsequent release of carbohydrates, the monolayer develops into a biofilm.
- Top of page
- Results and discussion
- Experimental procedures
In most natural environments, microbes are found attached to surfaces in communal structures known as biofilms. Associations between microbes and surfaces and among microbes in a biofilm may increase the availability of nutrients to certain members of the community and may also expand the community's repertoire of defences against environmental stresses (Hoyle et al., 1992; Bower and Daeschel, 1999; Yildiz and Schoolnik, 1999; Mah and O’Toole, 2001). Thus, biofilms may provide microbes with an adaptive advantage in the environment.
Genetic and microscopic studies of biofilm formation by Pseudomonas aeruginosa and other Gram-negative organisms have suggested that development of a mature, three-dimensional biofilm involves the following consecutive, discrete stages: (i) the planktonic stage (ii) the monolayer stage, and, finally (iii) the biofilm (Costerton, 1995; O’Toole et al., 2000; Watnick and Kolter, 2000; Danese et al., 2001). The planktonic stage consists of free-swimming bacteria. These bacteria carry appendages such as flagella or pili that may accelerate biofilm development (Pratt and Kolter, 1998; Kachlany et al., 2001; Sauer and Camper, 2001; Kang et al., 2002; Bechet and Blondeau, 2003; Di Martino et al., 2003). It should be noted, however, that many Gram-positive and Gram-negative bacteria are able to form biofilms in the absence of pili and flagella (McKenney et al., 1998; Watnick and Kolter, 1999; Toledo-Arana et al., 2001; Watnick et al., 2001; Klausen et al., 2003; Reisner et al., 2003). Thus, these appendages are not absolutely required for surface attachment.
The bacterial monolayer is formed by single cells that initially associate with the surface transiently and then become permanently immobilized. Very few genetic studies of the wild-type bacterial monolayer exist. This is because the wild-type bacterial monolayer is an intermediate stage in biofilm development and therefore difficult to isolate. The monolayer state has been isolated by mutagenesis, however. In P. fluorescens, a secreted protein has been identified that is required for the transition from transient to permanent attachment (Hinsa et al., 2003). Furthermore, in several Gram-negative organisms including V. cholerae and P. aeruginosa, mutations that arrest biofilm development in the monolayer state have been identified. In LB broth, mutation of genes involved in exopolysaccharide synthesis arrests V. cholerae cells in the monolayer stage of development (Watnick and Kolter, 1999). Mutation of genes involved in type IV pilus biogenesis arrest P. aeruginosa in the monolayer state (O’Toole and Kolter, 1998). Because of the paucity of monolayer-associated cells, quantification of gene transcription in this state is a challenge.
The final stage in biofilm development is defined by the formation of a highly organized, three-dimensional structure composed of bacterial pillars surrounded by water channels through which nutrients and waste products may be transported (Costerton et al., 1995; Kolter and Losick, 1998). Although the biofilm itself is heterogeneous, some general genetic and transcriptional features of the biofilm stage have been observed. Exopolysaccharide is required for the three-dimensional structure of most types of biofilms (Bonet et al., 1993; Allison et al., 1998; Watnick and Kolter, 1999; Danese et al., 2000). Furthermore, gene transcription in the biofilm is distinct from that in planktonic cells (Sauer and Camper, 2001; Whiteley et al., 2001; Tremoulet et al., 2002; Wen and Burne, 2002; Yoshida and Kuramitsu, 2002; Sauer, 2003; Schembri et al., 2003; Stanley et al., 2003). Genes required for the synthesis of flagella and pili, which are required for motility and surface attachment, are repressed in the biofilms of many Gram-negative bacteria (Sauer and Camper, 2001; Whiteley et al., 2001; Sauer et al., 2002; Schembri et al., 2003). These structures are only required during the initial stages of biofilm development and have the potential to destabilize the mature biofilm. In contrast, exopolysaccharide synthesis genes, which are crucial to the maintenance of the biofilm structure, show increased transcription in biofilm cells. Thus, progression to the biofilm state from the planktonic state requires a change in the gene transcription, the extracellular matrix composition, and the three-dimensional structure of the bacterial population.
Vibrio cholerae is both a natural inhabitant of aquatic environments and the intestinal pathogen that causes cholera (Colwell and Spira, 1992; Colwell and Huq, 1994; Colwell, 1996). It has been found in diverse marine, estuarine and fresh water environments in association with zooplankton, phytoplankton, crustaceans, insects and plants (Huq et al., 1983; 1986; Islam et al., 1989; Tamplin et al., 1990; Shukla et al., 1995; Castro-Rosas and Escartin, 2002). Adhesion to surfaces during infection of the human intestine and colonization of the aquatic environment may play an important role in the success of V. cholerae as a pathogen and an environmental organism. The toxin co-regulated pilus (TCP), a type IV bundle forming pilus, is primarily responsible for colonization of the mammalian intestine by V. cholerae (Taylor et al., 1987; Herrington et al., 1988; Thelin and Taylor, 1996; Tacket et al., 1998). In contrast, in LB broth, attachment of V. cholerae O1 El Tor to abiotic surfaces is accelerated by the mannose-sensitive haemagglutinin type IV pilus (MSHA) and the polar flagellum (Watnick et al., 1999; Chiavelli et al., 2001). Interestingly, the dependence of V. cholerae biofilm development on MSHA and the flagellum appears to be strain-dependent (Watnick and Kolter, 1999; Watnick et al., 2001). This suggests that V. cholerae produces a third mediator of initial attachment whose regulation varies among strains. In all V. cholerae strains studied, formation of a three-dimensional biofilm structure requires the vps genes, which encode enzymes involved in the synthesis of VPS, an exopolysaccharide found in the biofilm matrix (Wai et al., 1998; Mizunoe et al., 1999; Watnick and Kolter, 1999; Yildiz and Schoolnik, 1999). Regulation of VPS synthesis is complex. vps transcription, VPS synthesis, and biofilm development are activated by many environmental signals including (i) addition of monosaccharides to the growth medium and (ii) incorporation into a biofilm (Haugo and Watnick, 2002; Kierek and Watnick, 2003). Repressors of vps transcription, VPS synthesis and biofilm development have also been identified. High cell densities repress VPS synthesis and biofilm development through the action of HapR (Zhu et al., 2002; Hammer and Bassler, 2003; Vance et al., 2003; Zhu and Mekalanos, 2003). This suggests that quorum sensing acts to limit accumulation of cells on a surface. The flagellum is also likely to be a repressor of biofilm development as flagellar mutants display inappropriate VPS synthesis (Watnick et al., 2001). This appears to be a common theme as inverse regulation of exopolysaccharide and flagellum synthesis has been observed in other organisms (Burdman et al., 1998; Garrett et al., 1999). Lastly, CytR, a repressor of nucleoside uptake and catabolism, has been shown to repress vps transcription and decrease surface adhesion (Haugo and Watnick, 2002). VpsR, a σ54-dependent transcriptional activator, appears to be a central regulator of vps transcription through which many of the pathways controlling biofilm development pass (Yildiz et al., 2001). Strains harbouring engineered mutations in many of the pathways that repress VPS synthesis display a wrinkled or rugose colony morphology on LB-agar plates (Jobling and Holmes, 1997; Watnick et al., 2001; Haugo and Watnick, 2002; Hammer and Bassler, 2003; Zhu and Mekalanos, 2003). Increased VPS synthesis has been demonstrated in these colonies. Spontaneous rugose strains also arise (White, 1938; Morris et al., 1996; Wai et al., 1998; Mizunoe et al., 1999; Yildiz and Schoolnik, 1999; Ali et al., 2002). It has been suggested that mutations in the hapR gene give rise to these rugose strains (Hammer and Bassler, 2003).
Biofilm formation has been described as a developmental process in which the bacterial cell evolves from a planktonic cell to a monolayer cell to a terminally differentiated biofilm cell (Costerton, 1995; Palmer and White, 1997; O’Toole et al., 2000; Watnick and Kolter, 2000; Danese et al., 2001). Whereas studies of the planktonic and biofilm stages of biofilm development have begun to provide insights into the genetic requirements and transcription profiles of the biofilm-associated cell, the monolayer stage has not been studied using these techniques. Furthermore, the monolayer has not been proven to be a developmental stage which is distinct from the planktonic and biofilm stages both in its structural requirements and in its profile of gene transcription.
In this work, we have developed a growth medium that allows us to arrest wild-type V. cholerae cells in the monolayer stage and study this monolayer independently from the biofilm. Using this medium, we show that monolayer cells have genetic requirements and transcription profiles that are distinct from both planktonic and biofilm-associated cells.