Roberto Kolter

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar‐mediated motility and (ii) mutants defective in biogenesis of the polar‐localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase‐contrast microscopy. The wild‐type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time‐lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild‐type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non‐motile strain (carrying a mutation in flgK ) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell‐to‐surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

Populations of surface‐attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P. fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective (sad ). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.

Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient‐dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One‐half of these mutations was found to perturb normal flagellar function. Using defined fli, flh, mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose‐specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.

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

Fruiting body formation by Bacillus subtilis.

Spore formation by the bacterium Bacillus subtilis has long been studied as a model for cellular differentiation, but predominantly as a single cell. When analyzed within the context of highly structured, surface-associated communities (biofilms), spore formation was discovered to have heretofore unsuspected spatial organization. Initially, motile cells differentiated into aligned chains of attached cells that eventually produced aerial structures, or fruiting bodies, that served as preferential sites for sporulation. Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production. The formation of aerial structures was robust in natural isolates but not in laboratory strains, an indication that multicellularity has been lost during domestication of B. subtilis. Other microbial differentiation processes long thought to involve only single cells could display the spatial organization characteristic of multicellular organisms when studied with recent natural isolates.

Genetic approaches to study of biofilms.

Interest in the study of microbial biofilms has increased greatly in recent years due in large part to the profound impact biofilms have in clinical, industrial, and natural settings. Traditionally, the study of biofilms has been approached from an ecological or engineering perspective, using a combination of classical microbiology and advanced microscopy. We and others have begun to use genetic approaches to understand the development of these complex communities. To begin we must answer the question: What is a biofilm? This definition, by necessity, may be quite broad because it is clear that many organisms can attach to a variety of surfaces under diverse environmental conditions. Therefore, in the context of this article we will operationally define a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.

A role for excreted quinones in extracellular electron transfer

Respiratory processes in bacteria are remarkable because of their ability to use a variety of compounds, including insoluble minerals, as terminal electron acceptors. Although much is known about microbial electron transport to soluble electron acceptors, little is understood about electron transport to insoluble compounds such as ferric oxides. In anaerobic environments, humic substances can serve as electron acceptors and also as electron shuttles to ferric oxides. To explore this process, we identified mutants in Shewanella putrefaciens that are unable to respire on humic substances. Here we show that these mutants contain disruptions in a gene that is involved in the biosynthesis of menaquinone. During growth, the wild type releases a menaquinone-related redox-active small molecule into the medium that complements the mutants. This finding raises the possibility that electron transfer to a variety of oxidants, including poorly soluble minerals, may be mediated by microbially excreted quinones that have yet to be identified.

Steps in the development of a Vibrio cholerae El Tor biofilm

We report that, in a simple, static culture system, wild‐type Vibrio cholerae El Tor forms a three‐dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose‐sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three‐dimensional biofilm architecture.

Exopolysaccharide Production Is Required for Development of Escherichia coli K-12 Biofilm Architecture

Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of Escherichia coli K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.

d-Amino Acids Trigger Biofilm Disassembly

Biofilm Today, Gone Tomorrow Most bacteria can form complex, matrix-containing multicellular communities known as biofilms, which protect residents from environmental stresses such as antibiotic exposure. However, as biofilms age, nutrients become limiting and waste products accumulate, and biofim disassembly is triggered. Now Kolodkin-Gal et al. (p. 627) have found that d-amino acids found in conditioned medium from mature biofilms of Bacillus subtilis prevent biofilm formation and trigger existing biofilm disassembly. Bacteria secrete an unusual form of amino acids to escape from aging communities by dissolving the surrounding matrix. Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of d-leucine, d-methionine, d-tyrosine, and d-tryptophan that could act at nanomolar concentrations. d-Amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of d-Amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. d-Amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. d-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.