Inactivation of sagS and bfiS arrested biofilm formation prior to the maturation-1 stage of biofilm development, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Discontinuation of sagS

The goal of this proposal is to identify biofilm-specific regulatory proteins involved in the regulation of initial biofilm formation that once inactivated, impair or arrest the biofilm formation process. We have identified 8 regulatory proteins which become sequentially phosphorylated/dephopshorylated during biofilm formation. Inactivation of sagS and bfiS arrested biofilm formation prior to the maturation-1 stage of biofilm development, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Discontinuation of sagS and bfiS expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. plkR inactivation did not affect biofilm formation while plkR overexpression coincided with dispersion. Downstream gene targets of selected regulators were identified. Inactivation of bfiS coincided with decreased virulence. Inactivation of sagS rendered biofilms as susceptible to antimicrobials as planktonic cells indicating that biofilm resistance is regulated. Findings from this research indicated biofilm formation to be governed by a previously unidentified regulatory program, and provided detailed information how to control or prevent biofilm growth by interrupting the sequence of gene activation and affecting a cell’s ability to attach to a surface and form deleterious biofilms. (a) Papers published in peer-reviewed journals (N/A for none) *Petrova, O.E., and K. Sauer. 2009. A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development. PLoS Pathogens 5: e1000668. ** Petrova, O.E., and K. Sauer. 2010. The novel two-component regulatory system BfiSR regulates biofilm development directly by controlling the small RNA rsmZ through CafA. J. Bacteriol. 192:5275-5288. * This publication received some media attention and excerpts were featured on the following websites: o http://www.sciencecodex.com/researchers_discover_new_ways_to_treat_chronic_infections o http://www.sciencedaily.com/releases/2009/12/091218151327.htm o http://www.healthjockey.com/2010/01/02/development-of-biofilms-may-lead-to-new-ways-for-treating-chronic-infections/ o http://www.britannica.com/bps/additionalcontent/18/47256242/A-Novel-Signaling-Network-Essential-for-Regulating-Pseudomonas-ae ruginosa-Biofilm-Development ** This publication was highlighted by a commentary in the Journal of Bacteriology (Goodman, 2010) as well by a journal highlight in the ASM journal Microbe (http://www.microbemagazine.org/index.php/10-2010-journal-highlights/2913-improved-understanding-of-regulation-of-biofilm-formation) List of papers submitted or published that acknowledge ARO support during this reporting period. List the papers, including journal references, in the following categories: (b) Papers published in non-peer-reviewed journals or in conference proceedings (N/A for none) 2.00 Number of Papers published in peer-reviewed journals: Number of Papers published in non peer-reviewed journals: (c) Presentations 0.00 Number of Presentations: 0.00 Non Peer-Reviewed Conference Proceeding publications (other than abstracts): Oral presentations, invited 09/2010 Regulatory systems required for biofilm development and the maintenance of the planktonic mode of growth. Biofilms IV, 4th International SFAM meeting on Biofilms, Winchester, UK. 07/2010 Biofilm Development and Dispersion. SFAM Summer conference, Brighton, UK. 04/2010 Signaling networks essential for regulating Pseudomonas aeruginosa biofilm development, University of Oklahoma Health Sciences Center, Oklahoma City, OK. 04/2010 Signaling networks essential for regulating Pseudomonas aeruginosa biofilm development, Department of Microbiology, Montana State University, Bozeman, MT. 03/2010 A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. Mathematical Biosciences Institute (MBI) workshop, Columbus, OH. 11/2009 A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. 5th ASM (American Society for Microbiology) Biofilms conference, Cancun, Mexico 10/2009 A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. Syracuse University, Syracuse, NY. 04/2009 Regulation of biofilm formation by Pseudomonas aeruginosa. Stevens Institute of Technology, Department of Engineering, Hoboken, NJ. 02/2009 Regulation of biofilm formation by Pseudomonas aeruginosa. University of Rochester, Department of Microbbiology and Immunology, Rochester, NY. 10/2008 Using tags and isotopes to track post-translational modifications during biofilm formation by Pseudomonas aeruginosa. Binghamton University, Department of Chemistry, Binghamton, NY. -----Poster presentations at national and international conferences 11/2009 * Petrova, O., K. Sauer. Novel regulatory systems responsible for maintaining the planktonic lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. 5th ASM (American Society for Microbiology) Biofilms conference, Cancun, Mexico 09/2009 ** Petrova, O., K. Sauer. Coordinated signaling events regulate transition from initial attachment to maturation during Pseudomonas aeruginosa biofilm formation. Eurobiofilms 2009, Rome, Italy. 05/2009 Petrova, O., K. Sauer. Pseudomonas aeruginosa biofilm development is coordinated by a sequential series of signal transduction events. ASM conference, 109th General meeting, Philadelphia, PA. 07/2008 Petrova, O., G. Vazquez, and K. Sauer.Pseudomonas aeruginosa biofilm development is coordinated by multiple stage-specific transcriptional regulators. Corning Incorporated Science & Technology, Elmira, NY. 05/2008 Petrova, O., G. Vazquez, and K. Sauer. Pseudomonas aeruginosa biofilm development is coordinated by multiple stage-specific transcriptional regulators. ASM conference, 108th General meeting, Boston, MA. Number of Non Peer-Reviewed Conference Proceeding publications (other than abstracts): 0 Peer-Reviewed Conference Proceeding publications (other than abstracts): (d) Manuscripts Number of Peer-Reviewed Conference Proceeding publications (other than abstracts): 0 Manuscripts in preparation Marques, C, O.E. Petrova, Y. Li, and K. Sauer. Antimicrobial resistance and virulence are developmental aspects of biofilm formation by the human pathogen Pseudomonas aeruginosa. Petrova, O.E., and K. Sauer. SagS contributes to the sessile-motile switch by the human pathogen Pseudomonas aeruginosa Petrova, O.E., and K. Sauer. Role of the transcriptional regulator PlkR in attachment and biofilm dispersion. Number of Manuscripts: 3.00