Cloning chiC from insecticidal Pseudomonas aeruginosa PAO1

Abstract

Chitinolytic microorganisms display potent insecticidal abilities, which has garnered significant attention in the agricultural industry due to the numerous challenges surrounding pest management. Chitin-degrading bacteria have stood out as a promising and sustainable biocontrol agent, which has motivated research efforts to understand the mechanisms that underlie bacterial insecticidal capabilities. Some bacteria can produce chitinase enzymes, which degrade chitin present in the exoskeletons of insects, ultimately leading to insect lethality. It has been recently discovered that chiC, the gene that encodes for the chitinolytic ChiC enzyme, is found in highly insecticidal species, including several pseudomonads. This includes Pseudomonas aeruginosa PAO1, which can secrete ChiC into the extracellular environment. In this study, we aimed to bioinformatically analyze ChiC and clone chiC from P. aeruginosa PAO1 into a plasmid vector. PCR was used to amplify chiC from the genome of P. aeruginosa PAO1. The PCR product was subcloned into vector pCR2.1, resulting in plasmid pGKMS21. Several clones of pGKMS21 were analyzed by Sanger sequencing and the presence of the full-length chiC gene was confirmed. Sequence alignment of the sequenced region from pGKMS21 showed that it had close to 100% identity with the chiC (NC_002516.2) from P. aeruginosa PAO1. The pGKMS21 construct serves as a foundational tool that can be used in future studies to isolate chiC for protein expression and purification studies, allowing for further in-depth characterization of ChiC.