Evaluating aroA gene essentiality and EPSP synthase vulnerability in Mycobacterium smegmatis under different nutritional conditions

The epidemiological importance of bacteria from the genus is indisputable and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important metabolites in bacteria, represents a target for inhibitors of growth. The -encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene knockout, gene knockdown and kinetic assays to evaluate gene essentiality and the vulnerability of its protein product, EPSPS synthase from (EPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality of EPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions of gene, together with kinetic assays using WT and mutant recombinant proteins, we show that gene essentiality depends on EPSPS activity. To evaluate EPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces for gene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating that EPSPS vulnerability depends on the environment conditions.