Controlling Biofilm Formation and Dispersal

Almost all bacteria grow on solid surfaces or at air/liquid interfaces in complex communities known as biofilms. These communities are useful for engineering applications like remediation, biocorrosion, biocatalysis, and microbial fuel cells and are important for medical problems such as chronic infections. We created the first engineered biofilm for any application by engineering a consortium to reduce the biocorrosion of 1018 mild steel and 304 stainless steel. Since it has only one membrane, Bacillus subtilis was engineered to secrete the human peptide antimicrobial bactenecin (12 aa) to inhibit the growth of the deleterious sulfate-reducing bacterium Desulfovibrio vulgaris; growth of the corrosion-causing D. vulgaris was reduced by 36 fold in the biofilm, and corrosion was reduced by 12-fold (J. Industr. Microbiol. Biotechnol. 22:167, 1999). This work is important in that it utilized a consortium rather than a single species, showed that bacterial populations could be controlled in biofilms, and showed that cells could be engineered to achieve an engineering aim. We also have investigated the genetic basis of biofilm formation using the best-studied bacterium, Escherichia coli, and discovered that the quorum-sensing compound autoinducer 2 increases biofilm formation (J. Bacteriol. 188: 305, 2006) and that indole is a QS signal that inhibits biofilm formation through the “orphan” QS regulator SdiA that E. coli uses to sense acylhomoserine lactones of other bacteria (BMC Microbiol. 7:42, 2007). We then used this to create the first the first synthetic circuit that utilizes a QS signal to control biofilm formation: a circuit was built in which the extracellular concentration of indole in a consortium containing E. coli and Pseudomonas fluorescens was decreased by cloning toluene o-monooxygenase into the chromosome of the pseudomonad. The 22-fold reduction in the extracellular concentration of the inhibitor indole led to an 12-fold increase in the biofilm formation of E. coli (BMC Microbiol. 7:42, 2007). This work is important as it showed that the level of biofilm formation of a specific member of a consortium may be controlled by controlling the concentration of a QS compound. Cementing the role of indole on E. coli biofilm formation and its relation to SdiA, protein engineering of SdiA was performed via error-prone PCR. Using this approach, 4457 colonies were screened for altered biofilm formation to identify a SdiA variant with amino acid replacements F7L, F59L, Y70C, M94K, and K153X that reduced biofilm formation by 20-fold due to an increase in indole concentration (Appl. Environ. Microbiol. 75:1703, 2009). Another SdiA variant was identified that has four amino acid replacements (E31G, Y42F, R116H, and L165Q) that led to 7-fold higher biofilm formation in the presence of N-octanoyl-D,L-homoserine lactone and N-(3-oxododecatanoyl)-L-homoserine lactone produced by pseudomonads; this activity is opposite that of wild-type SdiA which reduces biofilm formation in the presence of AHL signals. This work was important since it demonstrated the power of protein engineering for creating tools to control biofilm formation in that the regulator SdiA was converted into both a more sensitive inhibitor of biofilm formation with indole as well as its activity was reversed as it was converted into a stimulator of biofilm formation in the presence of signals produced by other bacteria. We have also engineered the first global regulator to control biofilm formation, H-NS of E. coli. H-NS is a global regulator that represses transcription by recognizing curved DNA sequences and is widely conserved in Gram negative bacteria. Using protein engineering (error-prone PCR), 2104 mutants were screened for altered biofilm formation, and H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared to wild-type H-NS. Hence, the activity was flipped in that wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it. The mechanism by which H-NS K57N reduces biofilm formation is through its interaction with the nucleoid-associated proteins Cnu and StpA which leads to enhanced the excision of one of nine resident cryptic prophage in E. coli, the defective prophage Rac (wild-type H-NS represses excision). Excision of the prophage results in cell lysis through the expression of toxin HokD (Microb. Biotechnol. 3:344,2010). This work is important in that it demonstrates that a global regulatory system may be evolved through a single amino acid change in its N-terminal oligomerization domain to control biofilm formation, prophage excision, and apoptosis. Arguably one of the loftiest goals related to biofilm formation is to be able to remove existing biofilms, either to treat disease or to control biofilm formation for engineering applications. We have utilized two protein engineering approaches to perform the first directed evolution of proteins to get biofilms to disperse: one involves the global regulator Hha and is not based on the second messenger c-diGMP and the other relies on a novel c-di-GMP-binding protein, BdcA, that titrates c-di-GMP and thereby causes dispersal. Hha regulates 163 loci via it partner, H-NS, and has been related to biofilms as we have identified that hha is induced 30 fold in E. coli biofilms (Appl. Micro. Biotechnol. 64:515, 2004) and Hha decreases initial biofilm formation by repressing the transcription of rare codon tRNAs and by repressing transcription of fimbrial genes (PLoS ONE 3:e2394, 2008). Hha is also toxic and leads to cell lysis and biofilm dispersal due to activation of prophage lytic genes and due to the induction of protease ClpXP. Hence, Hha is a global transcriptional regulator related to biofilm formation; therefore, we reasoned that it could be engineered to control biofilm dispersal. Using random mutagenesis of hha and a direct screen for biofilm dispersal after 32 hr (which includes induction of hha for 8 h), Hha variant Hha13D6 (D22V, L40R, V42I, and D48A) was obtained that causes nearly complete biofilm dispersal (96%) by increasing apoptosis. Importantly, Hha13D6 induced dispersal without affecting initial biofilm formation. Hence, global regulator Hha may be evolved to cause biofilm dispersal in a manner distinct from initial biofilm formation. Unlike Hha13D6, BdcA (biofilm dispersal via c-di-GMP, formerly YjgI), was engineered to cause biofilm dispersal by decreasing the concentration of second messenger c-di-GMP. To identify BdcA, a biofilm screen was conducted of uncharacterized genes related to the protein we discovered that exports the bacterial signal autoinducer 2, TqsA (J Bacteriol 188:587, 2006), and the bdcA mutant was found to be deficient in biofilm dispersal. Hence, BdcA must positively regulate biofilm dispersal. Since BdcA increased motility and extracellular DNA production while decreasing exopolysaccharide, cell length, and aggregation, it was deduced that BdcA decreases the concentration of the intracellular messenger c-di-GMP. 31P nuclear magnetic resonance spectroscopy was used to show that BdcA binds c-di-GMP but does not act as a phosphodiesterase. To increase biofilm dispersal, protein engineering was used to evolve BdcA for greater c-di-GMP binding. A novel screen was developed that relies on the increase in swimming motility that occurs as c-di-GMP levels are decreased. The best BdcA variant had the single amino acid change E50Q that causes nearly complete removal of biofilms in flow cells without affecting initial biofilm formation. Hence a second genetic switch was created that may be used to remove biofilms after they are formed. This work is important as it identified a completely new way to control c-di-GMP concentrations (a new protein was identified), and it demonstrated that a ubiquitous second messenger could be controlled to create a general method to control biofilm dispersal. Therefore, we have conducted four sets of protein engineering to create proteins that allow us to control biofilm formation.