Half of all genes in the Pseudomonas aeruginosa genome have either no homology to any previously reported sequence or are homologues of previously reported genes of unknown function. The signature-tagged mutagenesis (STM) screening method allows to explore the role of these hypothetical and unknown proteins for the colonization and persistence of P. aeruginosa in eukaryotic hosts. A plasposon STM library was constructed in the virulent clinical P. aeruginosa isolate TBCF10839 that can persist in polymorphonuclear leukocytes (PMNs). The STM library was screened for plasposon mutants that were attenuated in the killing of the nematode Caenorhabditis elegans, deficient in quorum sensing and production of type II secretion effector proteins or had become more susceptible to killing by PMNs in phagocytosis assays. The three screens revealed in total 69 attenuated mutants. Fifteen mutants that carried the transposon in potential novel virulence determinants of yet unknown function were selected for further analysis. The mutants were characterized in their transcriptome and proteome and their cytotoxicity in vitro and their virulence in worm and mouse infection models in vivo. Previous studies had revealed a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins. Testing of our novel targets did not reveal such a strict conservation. The functional characterization revealed that the fifteen proteins play highly diverse roles in the cell and become habitat-specific virulence factors upon exposure to specific hosts and/or upon exposure to specific stress conditions or host defense mechanisms.