U T E R Ö M L I N G

Multicellular communities of single-celled organisms attached to a surface are the predominant form of life on

Earth. Development of these biofilms involves distinct stages of self-organization, starting with a single cell that senses and approaches a sur-face. Numerous factors have been identified that affect the formation, structure, metabolism and regulation of biofilms at the population level, but their formation has rarely been quantita-tively investigated at the single-cell level. In a paper published on Nature’s website today, Zhao et al.1 track the fate of individual bacte-rial cells from reversible surface attachment to microcolony formation. They show that the early stages of cell-to-surface contact are highly dynamic, with the bacteria exploring the surface and priming their environment for subsequent biofilm development.

During the early stages of biofilm formation2, motile bacteria approach a surface using a swimming action mediated by flagella — heli-cal rotatory filaments that are more than twice the cell’s length. On sensing a surface, a tighter, although reversible, contact is established through retractable appendages such as pili or fimbriae, which support surface crawling and contribute to the shaping of the three-dimensional architecture of biofilms3. Sur-face contacts mediated through these cellular extensions subsequently trigger the bacteria to secrete adhesive extracellular-matrix compo-nents4,5, which mediate irreversible binding, a prerequisite for the development of micro-colonies on biotic and abiotic surfaces. These microcolonies (clusters of up to 50 cells) are the foundation of the mature three-dimen-sional biofilm, which has distinct physiological properties, including antibiotic resistance and immune-system resilience.

But exactly how surface contact is translated into microcolony formation is not fully understood. To address this question, Zhao et al. explored the fate of individual cells of Pseudomonas aeruginosa, an environmental bacterium and formidable pathogen. They

tracked hundreds of thousands of cells in a defined area in time and space using a mas-sively parallel cell-tracking algorithm6. The authors found that the cells used type IV pili to explore the surface immediately after initial contact. Surprisingly, the bacteria did this in a non-random manner: a significant area of the surface was never visited, many places were visited once, and a few locations were visited more than 100 times.

To investigate this unexpected result, Zhao

and colleagues focused on the role of Psl, the adhesive polysaccharide secreted by P. aer-uginosa5. Their experiments revealed that the bacteria leave a trail of Psl as they move along the surface, but not of extracellular DNA, another component required for the initial establishment of biofilms. The researchers also found that Psl was unequally distributed on the surface — the areas with highest Psl deposition corresponded to those that had been visited the longest by bacteria, through either multiple visits or longer visiting time, and these sites constituted the founding sites of microcolonies. The authors propose that the mediators of the unequal surface exploration by the bacteria are the type IV pili, and the fact that these are attracted to Psl-rich regions gen-erates a positive feedback loop.

To test this idea, Zhao et al. generated a mutant P. aeruginosa strain that lacked the gene encoding Psl. Although this mutant was unable to initiate irreversible attachment and microcolony formation, the cells still moved along the surface, albeit with significantly shorter contact times (Fig. 1a). Taking the total

M I C R O B I O L O G Y

Bacterial communities as capitalist economies Tracking the behaviour of bacteria as they group together on a surface reveals a ‘rich-get-richer’ mechanism in which polysaccharide deposition and cellular location amplify in a positive feedback loop.

a

b

c

Type IV pili

Movement speed

Flagellum

Psl

Amount ofPsl deposited

Bacteriallocalization

+

+

Figure 1 | A positive feed-forward circuit steers biofilm formation. Pseudomonas aeruginosa bacteria use retractable structures called type IV pili to crawl along a surface after initial contact. Zhao and colleagues1 show that the cells’ exploration of the surface and subsequent development of a multicellular biofilm depends on their secretion of the adhesion molecule Psl, the presence of which promotes bacterial residence at a particular site. a, Mutant cells that produce no Psl explore a large surface area but are unable to irreversibly attach to it, such that no microcolonies form. b, Wild-type P. aeruginosa leave trails of Psl as they explore the surface. In a positive feedback loop, Psl deposition extends the cells’ sojourn time, such that they self-organize and form microcolonies at the sites of greatest Psl levels. The daughters of some cell divisions will remain, but because cells can still enter or leave the microcolony, it will contain several cell lineages. c, Bacteria that produce high amounts of Psl partake of less surface exploration, and a high proportion of daughter cells are retained at the location of cell division, leading to exponential growth of microcolonies founded by only one lineage.

| N A T U R E | 1

NEWS & VIEWSdoi:10.1038/nature12103

© 2013 Macmillan Publishers Limited. All rights reserved

number of cell-to-surface visits into account revealed that the mutant bacteria explored a larger surface area in a more random way than the wild-type cells. The authors then investi-gated a P. aeruginosa strain that produced higher levels of Psl than the wild-type bacte-rium: these cells spent yet more time in highly visited areas and even less time elsewhere.

Thus, it seems that the bacteria are guided by a synergistic ‘rich get richer’ mechanism, in which cells go where other cells go most often. The authors show that the bacterial surface-exploration behaviour could be described by a power law called Zipf ’s law — a probability distribution in which the frequency of an event decreases with a defined interval. In this case, the event is the visit frequency, and sites vis-ited only once are most numerous, sites visited twice are ranked next, and so on. Many self-organized systems, including wealth distribu-tion in capitalist economies, follow Zipf ’s law. From the authors’ results, it also follows that the frequency distribution of bacterial visits varies as a power of Psl secretion. Thus, Zhao and col-leagues suggest that, for the bacteria, this sys-tem results in some ‘elite’ bacteria being located at sites that are extremely rich in communally produced Psl, and that this social structure is required for microcolony formation.

The observed Psl-dependent movement also had a profound effect on microcolony com-position. Microcolonies formed by wild-type cells included several lineages because cells left and joined the area (Fig. 1b). By contrast, in Psl-overproducing cells, microcolonies

formed earlier and the bacterial cells were of one lineage, because mother and daughter cells tended to remain in the area (Fig. 1c).

This study and previous work from the same research group6 provide the most detailed picture yet of early biofilm forma-tion in P. aeruginosa. The present results also define the phase between reversible and irre-versible attachment — during which both motility and adhesion factors are expressed, enabling the bacteria to scan the surface of a potential new home and simultaneously prepare the home for settlement — as a dis-tinct developmental stage in biofilm forma-tion. It will be intriguing to see whether other biofilm-forming bacteria, such as Escherichia coli7 or Salmonella typhimurium8, connect sur-face movement and polysaccharide produc-tion in a similar way. It will also be interesting to assess how opposite processes — motility and attachment — are simultaneously regu-lated in a single cell. One obvious candidate to govern both processes is the ubiquitous bacte-rial signalling molecule cyclic dimeric GMP (c-di-GMP), which regulates the transition between motility and sessility9. Psl stimulates the production of c-di-GMP10 such that the priming of surfaces with Psl gradually directs cells towards sessility, but also exposes indi-vidual cells to different microenvironments. This can lead to variability in the population, including bistability.

Surface scanning leading to social self-organization is not unique to P. aeruginosa and the initiation of biofilm formation. The

gastrointestinal pathogen S. typhimurium, for example, uses flagella-mediated near-surface swimming for exploration of the surface of epi-thelial cells in the gut11. Here, the exploratory movement facilitates target-site selection and cooperative invasion by the bacteria as they infect these cells. Thus, it seems that the onset of biofilm formation and the infection pro-cess are governed by similar basic principles, suggesting that this behaviour may be a target for developing new ways to inhibit surface col-onization by bacteria or to prevent infection. ■

Ute Römling is in the Department of Microbiology, Cell and Tumor Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.e-mail: ute.romling@ki.se

1. Zhao, K. et al. Nature http://dx.doi.org/10.1038/nature12155 (2013).

2. O’Toole, G., Kaplan, H. B. & Kolter, R. Annu. Rev. Microbiol. 54, 49–79 (2000).

3. Barken, K. B. et al. Environ. Microbiol. 10, 2331–2343 (2008).

4. Li, G. et al. Mol. Microbiol. 83, 41–51 (2012).5. Mann, E. E. & Wozniak, D. J. FEMS Microbiol. Rev. 36,

893–916 (2012).6. Gibiansky, M. L. et al. Science 330, 197 (2010).7. Thomas, W. E., Nilsson, L. M., Forero, M., Sokurenko,

E. V. & Vogel, V. Mol. Microbiol. 53, 1545–1557 (2004).

8. Grantcharova, N., Peters, V., Monteiro, C., Zakikhany, K. & Römling, U. J. Bacteriol. 192, 456–466 (2010).

9. Pultz, I. S. et al. Mol. Microbiol. 86, 1424–1440 (2012).

10. Irie, Y. et al. Proc. Natl Acad. Sci. USA 109, 20632–20636 (2012).

11. Misselwitz, B. et al. PLoS Pathog. 8, e1002810 (2012).

2 | N A T U R E | © 2013 Macmillan Publishers Limited. All rights reserved