The opportunistic pathogen Pseudomonas aeruginosa utilizes a variety of virulence factors to infect a wide range of hosts. Virulence genes in this organism are many times controlled by two-component regulatory systems consisting of a sensor histidine kinase and a response regulator giving them high importance in research. Orphan response regulators consist of genes that have been proposed to be a response regulator but have not been studied to determine if they do work in a two-component regulatory system or not. Investigating these orphan response regulators could potentially lead to the finding of another regulator of virulence genes. Non-polar deletions were designed using splicing of genomic segments by overlapping extension. A variety of phenotypic assays, liquid-killing assays with the nematode C. elegans, and virulence assays with macrophages were utilized to determine if these orphans were different from the wild-type strain PAO1. If attenuated, these genes can be further studied to find new and novel regulators of virulence in Pseudomonas aeruginosa.