Analysis of secreted metabolites during predation of Myxococcus xanthus on Pseudomonas aeruginosa and Escherichia coli

Myxococcus xanthus is a Gram-negative facultatively predatory bacterium that exhibits collective social behavior. M. xanthus feeds on organic nutrients as well as on susceptible prey bacteria. The bacteria swarms over prey cells, lysing and consuming the nutrients released. Interestingly, some bacteria have evolved strategies to evade myxococcal predation. One such bacterium is Pseudomonas aeruginosa PAO1, which is an opportunistic human pathogen that uses quorum sensing to communicate to grow, which can be detrimental to humans with compromised immune systems. Strains of PAO1 are multi-drug resistant, and thereby pose a significant therapeutic challenge. We first performed side-by-side spot predation assay on partial starvation media with M. xanthus and P. aeruginosa PAO1 on bilayer agar plates, with a porous cellophane layer separating the two layers. After a 48-hr. predation, the samples were extracted and placed into a 60:40 ratio of methanol and water mixture to be analyzed using Liquid Chromatography Mass Spectrometry (LCMS) to determine the role of secreted metabolites in the predation evasion response of PAO1. A second side-by-side spot predation assay was performed using M. xanthus and P. aeruginosa on partial starvation media as well. Circular agar blocks containing the spots were transferred to a special insert-cup porous base, containing sterile partial starvation liquid medium. Metabolites secreted will diffused through the agar into the porous base of the insert, excluding any bacteria traveling through. After 48-hr. of predation, the liquid medium containing the metabolites were labeled “cell free extracts” and used for analytical experiments. With use of these research approaches, we expect to see different expression of metabolites during predation of M. xanthus on either P. aeruginosa or E. coli and compare them to our predatory and prey controls. Determining the evasion strategies of this bacterium could lead to the development of alternative methods to combat this pathogen