..................................................................................................................................... 1 RESUMEN ...................................................................................................................................... 5 INTRODUCTION ........................................................................................................................... 11 1. BACTERIAL SENSING ........................................................................................................ 13 1.1. One-component systems ........................................................................................ 15 1.2. Two-component systems ........................................................................................ 15 1.3. Chemosensory pathways ........................................................................................ 16 1.3.1. The prototypal chemotaxis pathway (E. coli) .................................................. 18 1.3.2. More complex chemotaxis pathways: Pseudomonas ..................................... 19 1.3.3. Information available on Pseudomonas MCP ................................................. 22 2. BACTERIAL LIFESTYLE ...................................................................................................... 24 2.1. Biofilm in Pseudomonas .......................................................................................... 27 2.2. Chemosensory signaling pathways control biofilm formation and colonization. ... 29 3. CELL TO CELL COMMUNICATION IN BACTERIA ............................................................... 30 3.1. Quorum sensing ...................................................................................................... 30 3.1.1. QS in Pseudomonas aeruginosa ...................................................................... 32 3.2. LuxR-type proteins .................................................................................................. 35 3.2.1. Classes of LuxR-type proteins .......................................................................... 37 3.2.2. LuxR-type proteins oligomeric state ............................................................... 38 3.2.3. General model for LuxR-type proteins ............................................................ 39 3.3. Quorum quenching ................................................................................................. 40 3.3.1. Degrading bacterial QSS .................................................................................. 41 3.3.2. Interference with QSS receptor ...................................................................... 42 3.4. Inter-kingdom signaling ........................................................................................... 43 AIM OF THE THESIS AND SPECIFIC OBJECTIVES .......................................................................... 47 METHODOLOGY .......................................................................................................................... 51 Experimental Procedures ....................................................................................................... 53 Microbiologic and molecular biology techniques .............................................................. 53 Bioinformatics analysis....................................................................................................... 55 Protein purification ............................................................................................................. 56 Biophysical analysis ............................................................................................................ 57 Genotypic studies ............................................................................................................... 60 Gene expression studies ..................................................................................................... 61 Phenotypic studies .............................................................................................................. 62 Plant studies ....................................................................................................................... 65 RESULTS AND DISCUSSION .......................................................................................................... 67 CHAPTER 1 Assessment of the contribution of MCP-based signaling to biofilm formation ... 71 Brief introduction ................................................................................................................ 71 Results ................................................................................................................................. 74 Construction of bacterial MCP mutants and their analysis ............................................. 74 Identification of PP2249 as a chemotaxis receptor for Lamino acids ........................... 79 Identification of PP1228 as a polyamine chemotaxis receptor ...................................... 81 Influence of MCP mutations on competitive plant root colonization ............................ 85 Discussion ............................................................................................................................ 86 Supplementary material ...................................................................................................... 90 CHAPTER 2 Purification of functional recombinant LasR and RhlR of Pseudomonas aeruginosa from Escherichia coli cultures............................................................................. 105 Brief Introduction .............................................................................................................. 105 Results ............................................................................................................................... 107 LasR and RhlR can be produced as recombinant proteins in E. coli in the absence of added AHL ..................................................................................................................... 107 LasR and RhlR bind different acyl-homoserine lactones with similar affinity ............... 108 LasR and RhlR unfold at temperatures beyond 60 °C and AHL binding does not alter protein stability significantly ......................................................................................... 110 AHL binding does not alter the oligomeric state of RhlR and LasR ............................... 112 RhlR and LasR bind to promoter DNA ........................................................................... 114 RhlR stimulates transcription in vitro in a protein and AHL concentration dependent manner .......................................................................................................................... 115 Differential impact of AHL ligands on the RhlR mediated upregulation of gene expression ..................................................................................................................... 117 Discussion .......................................................................................................................... 119 Supplementary material .................................................................................................... 122 CHAPTER 3 Rosmarinic acid is a homoserine lactone mimic produced by plants that activates a bacterial quorum sensing regulator ................................................................................... 129 Brief introduction .............................................................................................................. 129 Results ............................................................................................................................... 131 Rosmarinic binds to purified RhlR with high affinity ..................................................... 131 Rosmarinic acid stimulates RhlR-mediated transcription ............................................. 133 Rosmarinic acid stimulates QS gene expression in vivo ................................................ 134 Rosmarinic acid increases biofilm formation, pyocyanin production, and elastase synthesis ........................................................................................................................ 139 Discussion .......................................................................................................................... 142 Supplementary material .................................................................................................... 144 CONCLUSIONS ........................................................................................................................... 147 CONCLUSIONES ......................................................................................................................... 153 BIBLIOGRAPHY ........................................................................................................................... 159 PUBLICATIONS ........................................................................................................................... 187