Pyrrolnitrin is integral for antimicrobial activity and phenazine biosynthesis of Pseudomonas chlororaphis strains

Pseudomonas species are recognized for producing a diverse array of microbial metabolites with significant potential across various fields. Pyrrolnitrin (PRN), a halogenated metabolite based on phenylpyrrole, exhibits potent antibiotic properties. This research aimed to examine the influence of pyrrolnitrin on the antagonistic properties of Pseudomonas chlororaphis strains PB-St2, FS2, and RP4. Mutants of P. chlororaphis were generated by inhibiting prnA using two distinct suicide vectors, pEX18Tc and pKC1132. Analysis via high performance liquid chromatography (HPLC) revealed that pyrrolnitrin production was completely eliminated in the pKC1132 mutant and decreased by 82.5% in the pEX18Tc mutant. Both mutants also exhibited reduced phenazine production, with pKC1132 mutants showing a 61.1% reduction in phenazine-1-carboxylic acid (PCA) and pEX18Tc-induced mutants displaying a 39.9% decrease in PCA. To further elucidate the dependence of pyrrolnitrin production on other regulatory elements, the complete prnABCD operon with its native promoter was cloned and expressed in Escherichia coli (BL21). The antimicrobial potential of purified pyrrolnitrin was evaluated against fungal plant pathogens, human bacterial pathogens, and cancer cell lines (HepG-2 and SF767). The most pronounced inhibitory effect on Alternaria alternata was observed with 100 µg of pyrrolnitrin, resulting in an 82% reduction in spore formation followed by its effect on Aspergillus niger, causing a 75% decrease in spore production. Pyrrolnitrin's antibacterial activity produced inhibition zones of 1.8 cm against Salmonella enterica, 3.4 cm against Bacillus cereus, and 1.4 cm against Staphylococcus sp. at a concentration of 75 µg. The antiproliferative effects of pyrrolnitrin on cancer cell lines demonstrated 50% inhibition of both HepG-2 and SF767 cell lines at concentrations of 15 µg and 25 µg, respectively. Pyrrolnitrin exhibited significant antifungal and antibacterial activities, as well as cytotoxic effects on cancer cell lines, indicating its potential as both a biocontrol agent and therapeutic compound.