Confocal Imaging of Cell-to-Cell Interactions Between Myxococcus xanthus and Pseudomonas aeruginosa PAO1
Disciplines
Bacteriology | Environmental Microbiology and Microbial Ecology | Integrative Biology
Abstract (300 words maximum)
Micropredators are a significant selective force driving the evolution of bacteria including human pathogens. Studies in our lab and beyond have shown that M. xanthus can influence the chemotactic behavior of Pseudomonas sp and other gram negatives such as Escherichia coli. We have tested the predatory ability of a laboratory reference strain of M. xanthus DK1622 against P. aeruginosa strain PAO1. We have found that live cells persist despite predation. The present study employs modified high-content time-lapse confocal microscopy of motile biofilm surfaces to provide a clearer picture of the activity of individual cells within a swarm. An eYFP-tagged M. xanthus strain DK1622 and P. aeruginosa were grown in liquid nutrient media at their respective optimal conditions, then washed in buffer, and concentrated to a final concentration of 2x107 cells per mL for Mixed Cell Predation Assays or final concentration of 2x109 cells per mL for M. xanthus 2x1010 cells per mL for P. aeruginosa for Side-by-Side Spot Predation Assays. Mixed Cell Predation Assays consisted of 2 μL volumes of predator and prey mixed by pipetting and spotted on a partial starvation media agar pad and allowed to dry. Spots were then observed for 2hr by confocal imaging. Side-by-Side Spot Predation Assays consisted of two 2 μL spots of predator and prey placed 1 mm apart on a partial starvation media agar pad and allowed to dry. Using the Zeiss LSM 900 Axio Observer Confocal Microscope, multiple time-lapse videos and images were taken simultaneously in a 12-well plate at various times of predation for a 48-hour period. The spots were observed for several days and Z-stacks were taken at the junction where the myxobacterial front met the Pseudomonas spot.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Ramya Rajagopalan
Confocal Imaging of Cell-to-Cell Interactions Between Myxococcus xanthus and Pseudomonas aeruginosa PAO1
Micropredators are a significant selective force driving the evolution of bacteria including human pathogens. Studies in our lab and beyond have shown that M. xanthus can influence the chemotactic behavior of Pseudomonas sp and other gram negatives such as Escherichia coli. We have tested the predatory ability of a laboratory reference strain of M. xanthus DK1622 against P. aeruginosa strain PAO1. We have found that live cells persist despite predation. The present study employs modified high-content time-lapse confocal microscopy of motile biofilm surfaces to provide a clearer picture of the activity of individual cells within a swarm. An eYFP-tagged M. xanthus strain DK1622 and P. aeruginosa were grown in liquid nutrient media at their respective optimal conditions, then washed in buffer, and concentrated to a final concentration of 2x107 cells per mL for Mixed Cell Predation Assays or final concentration of 2x109 cells per mL for M. xanthus 2x1010 cells per mL for P. aeruginosa for Side-by-Side Spot Predation Assays. Mixed Cell Predation Assays consisted of 2 μL volumes of predator and prey mixed by pipetting and spotted on a partial starvation media agar pad and allowed to dry. Spots were then observed for 2hr by confocal imaging. Side-by-Side Spot Predation Assays consisted of two 2 μL spots of predator and prey placed 1 mm apart on a partial starvation media agar pad and allowed to dry. Using the Zeiss LSM 900 Axio Observer Confocal Microscope, multiple time-lapse videos and images were taken simultaneously in a 12-well plate at various times of predation for a 48-hour period. The spots were observed for several days and Z-stacks were taken at the junction where the myxobacterial front met the Pseudomonas spot.