To build a biofilm.

Development is not restricted to so-called higher organisms. Developmental processes in bacteria include differentiation of a single cell, such as the swarmer-to-stalk transition by Caulobacter crescentus and spore formation by Bacillus subtilis. Other microbes, such as Myxococcus xanthus, produce specialized cells within a population to form sporulating fruiting bodies (19). As we learn more about microbial biofilm formation, it is becoming clear that this is yet another example of a bacterial developmental process (4, 12). Like other developmental systems, building a biofilm requires a series of discrete and well-regulated steps. While the exact molecular mechanisms may differ from organism to organism, the stages of biofilm development appear to be conserved among a wide range of microbes. These stages include attachment of cells to a substrate, the growth and aggregation of cells into microcolonies, and the maturation and maintenance of architecture (3–5) (Fig. 1). In this issue of the Journal of Bacteriology, Finelli and colleagues at the Hospital for Sick Children in Toronto demonstrate the use of a new tool for further dissecting the development of bacterial biofilms (8). Bacterial developmental processes contribute to the success of a microbe in its environment. In the case of M. xanthus, development of the fruiting body includes a division of labor between sporulating cells, which give rise to the seeds of future generations, and the stalk cells, whose final act is to lift these spores above the substratum and aid in their dispersal. Biofilms also enable specialization of cells within a population, as was shown by Branda et al., who demonstrated spore-specific gene expression within localized regions of B. subtilis biofilms (1). Despite recent progress, it is apparent that we still have a great deal to learn about the mechanisms of bacterial biofilm construction and the interplay among phenotypically distinct subpopulations within these communities. Finelli and colleagues took advantage of a well-known technique called IVET (for “in vivo expression technology”) (10) and adapted the system to study genes expressed in a mature biofilm. Three new genes required for biofilm formation by Pseudomonas aeruginosa were identified by their application of this system, called IBET (for “in-biofilm expression technology”). A key element of the IBET system is a strict nutritional requirement to allow the selection of biofilm-expressed promoters that rescue this auxotrophy. These researchers looked for promoters whose expression rescued the adenine deficiency of a P. aeruginosa purKE mutant that grows in a biofilm but not on minimal agar medium lacking adenine. Their approach was validated by mutational analysis when they found that three of the five biofilm-expressed genes were required for biofilm formation but played no role in planktonic growth. This study represents the first time that such an approach has been used to study development in a microbial system. Like other developmental pathways, P. aeruginosa biofilm development is controlled by a number of different regulators, including LasR, RhlR, GacA, RpoS, Crc, and PvrR (6, 23; reviewed in reference 4). A new regulator can now be added to this list, encoded by the open reading frame designated PA3782 (based on the P. aeruginosa genome project, www .Pseudomonas.com), which appears to code for a transcriptional regulator of the AraC-XylS family. These transcription factors have been well studied as regulators of carbon metabolism and are also known to control virulence factor expression by numerous pathogens (7). While there is at least one example of a AraC-XylS family regulator participating in microbial developmental—the PA3782 homolog AdpA is required for the formation of aerial hyphae in Streptomyces griseus (11)—a role for this large family of transcription factors in microbial development may be underappreciated. Of the 800 potential family members identified as of 2002 (7), many of which have no known function (24), it would come as no surprise if at least some participated in the regulation of biofilm formation and/or other developmental pathways. One important problem facing biofilm researchers is trying to understand the changes in metabolism bacteria undergo as they adapt to life in these communities. The second gene required for biofilm development identified by Finelli and colleagues, PA3701, is predicted to encode an alcohol dehydrogenase or oxidoreductase. It is likely that this enzyme is necessary for the physiological adaptation of P. aeruginosa to the biofilm mode of life. This protein can be added to a long list of other enzymes involved in amino acid biosynthesis, carbon metabolism, and respiration that were previously identified by genomic and proteomic approaches as being expressed within the context of a biofilm (18, 26). While understanding the role that such enzymes play in biofilms is often difficult, and more often ignored, this information may be key to getting a firm grip on the changes microbes undergo as they adapt to life in a community. The third biofilm gene identified (PA0240, designated opdF) codes for a predicted porin based on its similarity to the OprD family of porins. Although the function of OpdF in biofilm formation remains to be elucidated, potential roles for this membrane protein include transport of signaling molecules, nutrients, or metabolic products. Alternatively, this protein may be needed to adapt to conditions particular to a biofilm, such as high osmolarity (15), or may serve as adhesin, as has been predicted for other surface structures, such flagella, pili, * Mailing address: Department of Microbiology and Immunology, Rm. 202, Vail Building, N. College St., Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1248. Fax: (603) 650-1318. E-mail: [email protected].