Phenotypic MicroArray Screening of Neisseria gonorrhoeae in Chemically Defined Liquid Medium.

Abstract

Phenotype MicroArrays (PMs) provide a considerable benefit to the evaluation of potential vaccine/drug targets and the assessment of hypothetical protein function. Nearly 2000 conditions can be screened relatively quickly either to search for phenotypes associated with the loss of a protein or to understand metabolic differences between closely related bacterial isolates. The fastidious organism Neisseria gonorrhoeae presents an experimental challenge for phenotypic screening due to its nutrient restrictions and its autolytic activity upon reaching the stationary phase of growth. These limitations can be mitigated by modulating screening parameters. In this chapter, we describe a technique optimized for the phenotypic screening of N. gonorrhoeae FA1090 and isogenic mutant strains. Inoculum size and culturing times have been adjusted for growth in chemically defined, protein-free Graver-Wade liquid medium in the 96-well microtiter plate format employed by the PMs. With the conditions presented, highly reproducible gonococcal growth is achieved, and autolysis prior to the experimental endpoint is minimized.