Spatial Periodicity of Escherichia coli K-12 Biofilm Microstructure Initiates during a Reversible, Polar Attachment Phase of Development and Requires the Polysaccharide Adhesin PGA

Surface-associated bacterial microbial communities known as biofilms have a profound impact on processes throughout the biosphere (5, 6), suggesting that mechanisms responsible for the design of their structure are likely to be of wide importance. Biofilm development includes stages of cell attachment, growth, differentiation, and dispersal (15, 21). In a previous study, we used fast Fourier transform (FFT) analysis to examine the cellular architecture or microstructure of Escherichia coli biofilm (1). These analyses revealed that a two-dimensional layer of attached cells exhibits distinct spatial periodicity, which persists within the body of the developing three-dimensional biofilm (1). This periodicity is formed independently of surface chemistry and is similar in wild-type strains and in a csrA mutant, which forms elevated amounts of biofilm (10, 11, 26, 27). Possible models for the origination of the spatial patterns were considered, including an activator-inhibitor or Turing model (1). Computer simulations have also suggested that an autoinhibitory process could guide the development of biofilm structure (4). In E. coli K-12, attachment to abiotic surfaces and cell-cell adhesion depends on surface organelles and the adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA) (9, 17, 26, 27). Attachment is also affected by physical factors, including shear force and gravity. Although in some cases shear may enhance cell adhesion (25), we observed that flow tended to remove adhering E. coli cells and created anisotropy in the observed attachment patterns (1). Gravitational settling of bacterial cells facilitates attachment and is widely used for the primary inoculation of biofilm support surfaces (2). However, under static conditions or at low flow rates, when the bacterial cells are allowed to settle freely, it can be difficult to distinguish between attached versus settled cells. Two major classes of bacterial cells are immediately apparent within a biofilm-forming population: unattached planktonic cells and attached cells. Studies dating back to the 1940s reveal that bacterial interaction with a surface often involves an initial reversible or temporary attachment, followed by irreversible or permanent attachment (reviewed in reference 24). Temporary attachments are reversible within a short period of time, within seconds or a few minutes, and are thought to afford the bacterium an opportunity to sample the environment before forming permanent attachments or releasing from the surface (discussed in reference 3). Permanently attached cells remain surface associated for at least tens of minutes and provide the foundation for further biofilm development. Although the molecular mechanisms responsible for these two forms of attachment remain to be defined, they were recently shown to be genetically distinguishable in Pseudomonas species. Mutants lacking lapA or sadB in Pseudomonas fluorescens and P. aeruginosa, respectively, were unable to undergo the transition from reversible to irreversible attachment (3, 8). The attachment force should be different for temporary versus permanently attached cells. In principle, sheer force can be used to distinguish strength of attachment, e.g., in a flow cell. Although bulk flow rate is easily measured, the actual speed of the flow and thus the shear force may vary significantly within the chamber. Unlike shear force, gravitational field is homogeneous and affects all bacterial cells equally. With this idea, we developed a quantitative assay to distinguish cells according to their strength of attachment by monitoring their displacement by gravitational field. The gravity displacement assay revealed two classes of surface attachment: temporary and permanent. As shown for Pseudomonas species (3, 8), temporary attachment was mainly by a cell pole, whereas permanent attachment was mainly by the lateral cell surface. A csrA mutant of E. coli (reviewed in reference 18) formed an increased proportion of permanently attached cells and a new subclass of cells that were permanently attached via a cell pole. The major role of CsrA in biofilm formation is posttranscriptional repression of pgaABCD expression, which is needed for PGA synthesis (28). Mutants lacking the glycosyltransferase that synthesizes PGA (ΔpgaC) still formed polar attachments but did not undergo the transition to permanent attachment and formed aperiodic or quasirandom attachment patterns. In contrast, polar attachments were not observed for lipopolysaccharide mutants (waaG), which formed permanently attached cells and cell clusters that exhibited quasirandom FFT patterns. Thus, waaG mutants appear to bypass the temporary attachment step in development. Gravity displacement was reversible; a displaced population was able to rapidly reestablish temporary cell attachments possessing spatial periodicity. These findings suggest that the decision to remain temporarily attached or return to the planktonic phase is a pivotal step in E. coli K-12 biofilm formation, designed to promote the formation of spatial periodicity within the developing microstructure, and that PGA synthesis is integral to this process.