Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in a microfluidic device

To utilize biofilms for chemical transformations in biorefineries they need to be controlled and replaced. Previously, we engineered the global regulator Hha and cyclic diguanylate-binding BdcA to create proteins that enable biofilm dispersal. Here we report a biofilm circuit that utilizes these two dispersal proteins along with a population-driven quorum-sensing switch. With this synthetic circuit, in a novel microfluidic device, we form an initial colonizer biofilm, introduce a second cell type (dispersers) into this existing biofilm, form a robust dual-species biofilm and displace the initial colonizer cells in the biofilm with an extracellular signal from the disperser cells. We also remove the disperser biofilm with a chemically induced switch, and the consortial population could tune. Therefore, for the first time, cells have been engineered that are able to displace an existing biofilm and then be removed on command allowing one to control consortial biofilm formation for various applications.

[1]  James J. Collins,et al.  Dispersing biofilms with engineered enzymatic bacteriophage , 2007, Proceedings of the National Academy of Sciences.

[2]  D. Weibel,et al.  Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth. , 2011, Journal of the American Chemical Society.

[3]  A. Rickard,et al.  Biofilms and Biocomplexity , 2007 .

[4]  Woo Y. Lee,et al.  Microfluidic devices for studying growth and detachment of Staphylococcus epidermidis biofilms , 2008, Biomedical microdevices.

[5]  A. Kirkham,et al.  Pseudomonas aeruginosa Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis , 2002, Infection and Immunity.

[6]  A. Jayaraman,et al.  Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions. , 2010, Lab on a chip.

[7]  Thomas K. Wood,et al.  Reconfiguring the Quorum-Sensing Regulator SdiA of Escherichia coli To Control Biofilm Formation via Indole and N-Acylhomoserine Lactones , 2009, Applied and Environmental Microbiology.

[8]  P. Fu,et al.  A perspective of synthetic biology: Assembling building blocks for novel functions , 2006, Biotechnology journal.

[9]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[10]  M A Henson,et al.  Design and mathematical modelling of a synthetic symbiotic ecosystem. , 2008, IET systems biology.

[11]  Roberto Kolter,et al.  d-Amino Acids Trigger Biofilm Disassembly , 2010, Science.

[12]  N. Godessart,et al.  Chromosomal mutations that increase the production of a plasmid-encoded haemolysin in Escherichia coli. , 1988, Journal of general microbiology.

[13]  Mat E. Barnet,et al.  A synthetic Escherichia coli predator–prey ecosystem , 2008, Molecular systems biology.

[14]  H. Ceri,et al.  Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system. , 2001, FEMS microbiology letters.

[15]  Qun Ma,et al.  Engineering a novel c-di-GMP-binding protein for biofilm dispersal. , 2011, Environmental microbiology.

[16]  Thomas K. Wood,et al.  Engineering global regulator Hha of Escherichia coli to control biofilm dispersal , 2010, Microbial biotechnology.

[17]  Sara Hooshangi,et al.  From unicellular properties to multicellular behavior: bacteria quorum sensing circuitry and applications. , 2008, Current opinion in biotechnology.

[18]  J. Kaplan,et al.  Biofilm Dispersal: Mechanisms, Clinical Implications, and Potential Therapeutic Uses , 2010, Journal of dental research.

[19]  P. Seed,et al.  Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa , 1997, Journal of bacteriology.

[20]  Thomas K. Wood,et al.  Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H‐NS of Escherichia coli , 2010, Microbial biotechnology.

[21]  Carolyn G. Conant,et al.  New Device for High-Throughput Viability Screening of Flow Biofilms , 2010, Applied and Environmental Microbiology.

[22]  D. Davies,et al.  A Fatty Acid Messenger Is Responsible for Inducing Dispersion in Microbial Biofilms , 2008, Journal of bacteriology.

[23]  Blaise R. Boles,et al.  agr-Mediated Dispersal of Staphylococcus aureus Biofilms , 2008, PLoS pathogens.

[24]  J. Collins,et al.  Programmable cells: interfacing natural and engineered gene networks. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Claudia Schmidt-Dannert,et al.  Applications of quorum sensing in biotechnology , 2010, Applied Microbiology and Biotechnology.

[26]  B. Iglewski,et al.  Active Efflux and Diffusion Are Involved in Transport of Pseudomonas aeruginosa Cell-to-Cell Signals , 1999, Journal of bacteriology.

[27]  M. Cámara,et al.  Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. , 2009, Current opinion in microbiology.

[28]  Frances H. Arnold,et al.  Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium , 2011, PloS one.

[29]  L. Zeef,et al.  Characterization of Nutrient-Induced Dispersion in Pseudomonas aeruginosa PAO1 Biofilm , 2004, Journal of bacteriology.

[30]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[31]  V. Paul,et al.  Mini-review: quorum sensing in the marine environment and its relationship to biofouling , 2009, Biofouling.

[32]  R. Weiss,et al.  Programmed population control by cell–cell communication and regulated killing , 2004, Nature.

[33]  A. Jayaraman,et al.  Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin , 1999, Journal of Industrial Microbiology and Biotechnology.

[34]  F. Arnold,et al.  Engineering microbial consortia: a new frontier in synthetic biology. , 2008, Trends in biotechnology.

[35]  J. M. Dow,et al.  Diffusible signals and interspecies communication in bacteria. , 2008, Microbiology.

[36]  B. Ersbøll,et al.  Quantification of biofilm structures by the novel computer program COMSTAT. , 2000, Microbiology.

[37]  L. Tsimring,et al.  A synchronized quorum of genetic clocks , 2009, Nature.

[38]  E. Greenberg,et al.  Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Wolfe,et al.  A Complex Transcription Network Controls the Early Stages of Biofilm Development by Escherichia coli , 2006, Journal of bacteriology.

[40]  Seok Hoon Hong,et al.  Engineering biofilm formation and dispersal. , 2011, Trends in biotechnology.

[41]  Qun Ma,et al.  Engineering a novel cdi-GMP-binding protein for biofilm dispersalemi , 2011 .

[42]  Jan Sunner,et al.  Biocorrosion: towards understanding interactions between biofilms and metals. , 2004, Current opinion in biotechnology.

[43]  S. Kjelleberg,et al.  A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. , 2000, Environmental microbiology.

[44]  S. Molin,et al.  Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration. , 2001, Microbiology.

[45]  J. L. Pozo,et al.  The Challenge of Treating Biofilm‐associated Bacterial Infections , 2007, Clinical pharmacology and therapeutics.

[46]  Blaise R. Boles,et al.  Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms , 2005, Molecular microbiology.

[47]  Priscilla E. M. Purnick,et al.  The second wave of synthetic biology: from modules to systems , 2009, Nature Reviews Molecular Cell Biology.

[48]  H. Flemming,et al.  The biofilm matrix , 2010, Nature Reviews Microbiology.

[49]  Arul Jayaraman,et al.  Indole is an inter-species biofilm signal mediated by SdiA , 2007, BMC Microbiology.

[50]  Thomas K. Wood,et al.  Protein Translation and Cell Death: The Role of Rare tRNAs in Biofilm Formation and in Activating Dormant Phage Killer Genes , 2008, PloS one.

[51]  Ron Weiss,et al.  Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium , 2007, Proceedings of the National Academy of Sciences.

[52]  I. Ial,et al.  Nature Communications , 2010, Nature Cell Biology.

[53]  P. Seed,et al.  Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy , 1995, Journal of bacteriology.