Confirmation of Caspase-3-Like-Protease, Clp, in Pseudomonas aeruginosa as an Individually Regulated Gene and its Involvement in Healthy Colony Formation

Abstract

Pseudomonas aeruginosa is an important nosocomial opportunistic human pathogen. In other studies the caspase-3-like protein Clp was determined to be required for structured biofilm formation and nutrient limited growth. In this study, the clp: lux mutant phenotype previously observed was investigated by complementation analysis. Using bioinformatics we were able to identify a putative operon in which clp resides. A complementation analysis was performed to establish whether a clp mutation or a polar mutation caused the observed mutant phenotype. Colony morphology was observed by a Congo red assay. The clp:: lux mutant phenotype was characterized by colonies with numerous voids, frayed edges, reduced thickness and the absence of green pigmentation. The complemented mutant displayed restored wild type phenotype, flat colonies with spreading borders and green pigmentation. These observations verified that the complementation rescued the clp:: lux mutant, restoring the wild type Clp phenotype. Therefore, we concluded that the mutant phenotype was due to disruption of the clp gene, and not a polar mutation.