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Engineering Escherichia coli to Control Biofilm Formation, Dispersal, and Persister Cell Formation

Biofilms are formed in aquatic environments by the attachment of bacteria to submerged surfaces, to the air/liquid interface, and to each other. Although biofilms are associated with disease and biofouling, the robust nature of biofilms; for example, their ability to tolerate chemical and physical stresses, makes them attractive for beneficial biotechnology applications such as bioremediation and biofuels. Based on an understanding of diverse signals and regulatory networks during biofilm development, biofilms can be engineered for these applications by manipulating extracellular/intercellular signals and regulators.

Here, we rewired the global regulator H-NS of Escherichia coli to control biofilm formation using random protein engineering. H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared with wild-type H-NS (wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it) via its interaction with the nucleoid-associated proteins Cnu and StpA. H-NS K57N leads to enhanced excision of the defective prophage Rac and results in cell lysis through the activation of a host killing toxin HokD. We also engineered another global regulator, Hha, which interacts with H-NS, to disperse biofilms. Hha variant Hha13D6 was obtained that causes nearly complete biofilm dispersal by increasing cell death by the activation of proteases.

Bacterial quorum sensing (QS) systems are important components of a wide variety of engineered biological devices, since autoinducers are useful as input signals because they are small, diffuse freely in aqueous media, and are easily taken up by cells. To demonstrate that biofilms may be controlled for biotechnological applications such as biorefineries, we constructed a synthetic biofilm engineering circuit to manipulate biofilm formation. By using a population-driven QS switch based on the LasI/LasR system and biofilm dispersal proteins Hha13D6 and BdcAE50Q (disperses biofilms by titrating cyclic diguanylate), we displaced an existing biofilm and then removed the second biofilm.

Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Here, we engineered toxin MqsR with greater toxicity and demonstrated that the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.

Identiferoai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2011-12-10354
Date2011 December 1900
CreatorsHong, Seok Hoon
ContributorsWood, Thomas K., Jayaraman, Arul, Chen, Zhilei, Benedik, Michael J.
Source SetsTexas A and M University
Languageen_US
Detected LanguageEnglish
TypeThesis, thesis, text
Formatapplication/pdf

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