Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments. Currently, there is an explosive amount of biofilm research, most of it with the ultimate aims of its prevention, control, or eradication. By studying the genes that are differentially expressed in biofilms as well as those that are regulated by plant-derived biofilm inhibitors, we have begun to unravel how biofilms form and how to manipulate them; i.e., how to stimulate or inhibit them. Interestingly, we have found that E. coli and pseudomonads respond to signals they do not synthesize, that competition for signals is intense (to the extent that signals are manipulated), and that biofilm signals control pathogenicity loci.

Specifically, we have discovered that the cross-species, quorum-sensing signal autoinducer-2 (AI-2) induces biofilm formation 30-fold in E. coli K12 by increasing expression of 67 genes, primarily those associated with chemotaxis, motility, and flagellar synthesis including the specific motility loci qseBC and flhD (J. Bacteriol. 188:305, 2006). DNA microarrays also helped us to determine this induction of biofilms was via the completely uncharacterized protein B3022 (now MqsR) and that this protein was the master regulator of QseB. Through microarrays, we also discovered the protein which exports AI-2 (YdgG now TqsA) which has been theorized to exist but never found (J. Bacteriol. 188:587, 2006); deleting ydgG caused 31% of the bacterial chromosome to be differentially induced and 7.6% of the genes were repressed due to trapping AI-2 inside the cell. TqsA not only negatively modulates expression of flagella- and motility-related genes but also all the other known products essential for biofilm formation: 4 known operons for flagella synthesis and motility (flgABCDEFGHIJ, fliEFGHIJK, fliLMNOPQR, and motABcheAW), adhesion determinants (type 1 fimbriae and the autotransporter protein Ag43), curli production, colanic acid production, and production of b-1,6-N-acetyl-D-glucoseamine polysaccharide adhesin. Two other uncharacterized proteins were also identified that control quorum-sensing, YliH (now BssR) and YceP (now BssS); these two proteins help to regulate the indole and AI-2 response (Appl. Environ. Microbiol. 72:2449, 2006).

Since biofilm formation is dynamic, we analyzed temporal differential gene expression in an E. coli biofilm (Environ. Microbiol., in press, 2006) to discern additional proteins related to biofilm formation. AI-2 was found to influence mature biofilms and cold-shock proteins are positive biofilm regulators.

Using systems biology (a series of global transcriptome analyses, confocal microscopy, isogenic mutants, and dual-species biofilms), we have also determined that indole, the primary stationary phase product secreted by E. coli, is an interspecies biofilm signal (Appl. Environ. Microbiol. 70:2038, 2004 and submitted). It inhibits E. coli biofilms while stimulating those of pseudomonads. We found indole is a non-toxic signal that controls E. coli biofilms by inducing the sensor of the quorum sensing signal autoinducer-1 (SdiA) which in turn represses motility and influences acid resistance (e.g., hdeABD, gadABCEX). Isogenic mutants showed the associated proteins are directly related to biofilm formation, and SdiA-mediated transcription was shown to be influenced by indole. The reduction in motility due to indole addition results in the biofilm architecture changing from scattered towers to flat colonies. Additionally, there are 12-fold more E. coli cells in dual-species biofilms grown in the presence of Pseudomonas cells engineered to express toluene o-monooxygenase (TOM, which converts indole to an insoluble indigoid) than in biofilms with pseudomonads that do not express TOM due to a 22-fold reduction in extracellular indole. Hence, indole is an interspecies signal that may be manipulated by oxygenases of another bacterium to control biofilm formation, and pseudomonads respond to signals they do not synthesize. Further evidence that the indole effects are mediated by SdiA and AI-1 quorum sensing is that the addition of N-butyryl-, N-hexanoyl-, and N-octanoyl-L-homoserine lactones repress E. coli biofilm formation in the wild-type strain but not with the sdiA mutant; therefore, E. coli changes its biofilm in response to signals it cannot synthesize. The indole microarrays were also used to discover the uncharacterized protein YmgB mediates acid-resistance (renamed as AriR for regulator of acid resistance influenced by indole), and to discern that indole reduces acid resistance.

There are few known natural compounds which inhibit biofilm formation while not affecting cell growth, but the quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine alga Delisea pulchra inhibits biofilm formation in E. coli without inhibiting its growth by masking the quorum sensing signal AI-2 (Biotechnol. Bioengr. 88:630, 2004). By screening 13,000 plant extracts, another three potent biofilm inhibitors have also been found, including ursolic acid (Appl. Environ. Microbiol. 71:4022, 2005), and DNA microarrays have been used to gain insights into how these compounds function as well as to how biofilms may be controlled. Furthermore, we theorized that if indole were present at as high as 600 µM in cell supernatants and if the human gut has over 500 different species of bacteria, then bacteria would manipulate this quorum signal indole for their own ends, and that they would use relatively non-specific oxygenases to form oxidated indoles. By screening hydroxy indoles derived from monooxygenases, we have confirmed this hypothesis by finding an even more potent inhibitor of biofilms, 7-hydroxyindole. Evidence that these quorum signals control pathogenicity genes will also be provided as well as results related to protein engineering to re-wire the biofilm circuits.

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