Biofilm, city of microbes.

In most natural environments, association with a surface in a structure known as a biofilm is the prevailing microbial lifestyle. Surface association is an efficient means of lingering in a favorable microenvironment rather than being swept away by the current. Taken to the extreme, we may view the planktonic or free-swimming microbial phase primarily as a mechanism for translocation from one surface to another. Genetic studies of single-species biofilms have shown that they form in multiple steps (46), require intercellular signalling (7), and demonstrate a profile of gene transcription that is distinct from that of planktonic cells (35). From this perspective, biofilm formation may be viewed as a developmental process that shares some of the features of other bacterial developmental processes such as sporulation of gram-positive bacteria (9), fruiting body formation in Myxococcus xanthus (33, 40, 44), and stalked-cell formation by Caulobacter crescentus (13, 19, 24, 37, 48). In natural environments, however, the biofilm is almost invariably a multispecies microbial community harboring bacteria that stay and leave with purpose, share their genetic material at high rates, and fill distinct niches within the biofilm. Thus, the natural biofilm is less like a highly developed organism and more like a complex, highly differentiated, multicultural community much like our own city. There are several steps that we must take to optimize our lives in a city. The first is to choose the city in which we will live, then we must select the neighborhood in the city that best suits our needs, and finally we must make our home amongst the homes of many others. Occasionally, when life in the city sours, we leave. The same steps occur in the formation of a bacterial biofilm (Fig. 1). First, the bacterium approaches the surface so closely that motility is slowed. The bacterium may then form a transient association with the surface and/or other microbes previously attached to the surface. This transient association allows it to search for a place to settle down. When the bacterium forms a stable association as a member of a microcolony, it has chosen the neighborhood in which to live. Finally, the buildings go up as a three-dimensional biofilm is erected. Occasionally, the biofilm-associated bacteria detach from the biofilm matrix. Micrographs of these steps in biofilm formation by a single bacterial species are shown in Fig. 2. Although these micrographs are static views of the steps in biofilm formation, a biofilm is not a motionless heap of cells. Figure 3 shows the first frame of a real time movie, accessible at http//gasp.med.harvard.edu/biofilms/jbmini/movie.html, that documents the activity in a mature biofilm. In this frame, the pillars of a mature biofilm are visible, distributed on top of a monolayer of surface-associated cells. The associated movie shows that, in addition to fixed cells, there are motile cells that maintain their association with the biofilm for long periods of time, swimming between pillars of biofilm-associated bacteria. The biofilm, therefore, demonstrates a level of activity similar to that of a bustling city. The genetic basis of the steps in biofilm formation has been investigated for a number of bacterial species, including Escherichia coli (34), Pseudomonas aeruginosa (31) and Vibrio cholerae (46). For these studies, a simple genetic screen was utilized in which random transposon mutants are grown in 96-well plates (5, 16, 32). After removal of the planktonic cells, the remaining biofilm-associated cells are stained with crystal violet. Those wells with no crystal violet staining correspond to mutants that are defective in biofilm formation. These genetic screens for biofilm-defective mutants have shown that the initial interaction with the surface is accelerated by force-generating organelles such as type IV pili and flagella. Once temporary contact with the surface is made, bacteria use either flagella or type IV pili to move along the surface in two dimensions until other bacteria are encountered and microcolonies are formed or enlarged (31, 34, 46). Finally, exopolysaccharide production is necessary to stabilize the pillars of the biofilm (46). Competition studies between wild-type V. cholerae and pilus or flagellar mutants show that these structures provide a great advantage in surface colonization (P. I. Watnick and R. Kolter, unpublished results). Thus, speed of attachment may be an important factor in garnering an apartment in the microbial city. Evidence exists that different genes are transcribed in the planktonic and biofilm-associated phases of the bacterial life cycle. This is again reminiscent of a developmental process. Prigent-Combaret et al. performed a screen for genes in E. coli that are differentially expressed in biofilm-associated cells, using a library of random insertion mutants generated with a MudX transposon carrying a promoterless lacZ gene (35). One interesting finding from this study is that flagellin synthesis is decreased in biofilm-associated cells, while production of colanic acid, an exopolysaccharide made by E. coli, is increased. The situation appears to be similar in P. aeruginosa. Alginate is an exopolysaccharide that is found in P. aeruginosa biofilms (14). Transcription of algC, a gene involved in the production of alginate, is increased approximately fourfold in biofilmassociated cells as compared with planktonic cells (6, 15). Furthermore, for many years, researchers have noted that pulmonary isolates of P. aeruginosa are mucoid due to production of copious amounts of alginate (14). Recently, Garrett and coworkers noted that flagella are absent from these mucoid isolates (15). In addition, they showed by mutational analysis that while alginate synthesis is positively regulated by the alternative sigma factor s, this sigma factor negatively regulates the synthesis of the flagellum. This suggests that when synthesis of * Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1776. Fax: (617) 738-7664. E-mail: [email protected].