The plasmids expressing the different coloured AFPs were introduced into P. fluorescens by electroporation according to previous protocols [15]. The colony variants (WS and SCV) were derived from the Δ gacS strain which produces phenotypic variants when exposed to heavy metal stress [2]. Introduction of the plasmids had no observable effects on colony morphology. Biofilms were cultured in LB using the Calgary Biofilm Device (CBD) [16, 17], with shaking at 150 rpm, at 30℃ and approximately 95% relative humidity. A 1:30 dilution of a 1.0 McFarland standard

was prepared for each individual strain and the CBD was inoculated with either the individual strain or a 1:1 mixture of the two or three strains being co-cultured and then grown for the indicated time prior to imaging. Due to the extended growth times for this experiment (up to 96 h) viable cell counts JNK-IN-8 mw could not be obtained as the P. fluorescens variants grow very thick biofilms that could not be entirely removed by sonication. No new phenotypes were observed

eFT508 in vivo after 96 h of growth with any of the strains. Table 1 Strains and plasmids used in this study Strain or plasmid Description Source P. fluorescens CHA0 Wild-type strain [18] P. fluorescens CHA19 Contains a marker-less deletion of the gacS coding region [18] P. fluorescens SCV Small Colony Variant derived from the CHA19 strain [2] P. fluorescens WS Wrinkly Spreader derived from the CHA19 strain [2] pME6010 Rhizosphere stable plasmid, does not require antibiotic selection in P. fluorescens [19] pMP4655 pME6010 containing the coding sequence of see more enhanced GFP with the lac promoter [13] pMP4641 pME6010 containing the coding sequence of enhanced CFP Cytidine deaminase with the lac promoter [13] pMP4658 pME6010 containing the coding sequence

of enhanced YFP with the lac promoter [13] pMP4662 pME6010 containing the coding sequence of dsRed with the lac promoter [13] Microscopy and biofilm quantification Microscopy was performed according the protocols outlined previously [20]. The pegs were examined using a Leica DM IRE2 spectral confocal and multiphoton microscope with a Leica TCS SP2 acoustic optical beam splitter (AOBS) (Leica Microsystems). A 63 × water immersion objective used for all the imaging and the image capture was performed using Leica Confocal Software Lite (LCS Lite, Leica Microsystems). Imaging of the biofilms expressing the AFPs were obtained by breaking off a peg of the CBD and placing it on a coverslip with a drop of saline. Excitation/emission parameters for each of the AFPs were 488/500−600 for GFP, 514/525−600 for YFP, 458/465−600 for CFP, and 543/55−700 for dsRed. To reduce cross-talk between the different AFPs, images with more than one AFP were acquired sequentially by frame so only one AFP was being imaged at a time.