Construction of Pseudomonas aeruginosa strain with deletion of pyocyanin synthesis gene PAOII and study on its drug resistance

Objective To construct Pseudomonas aeruginosa (PA) strain with deletion of the pyocyanin synthesis gene PAOII and investigate its drug resistance.

Methods By analyzing the genome of the PAOII genotype strain of PA, the distribution of genes/gene clusters related to pyocyanin synthesis in the genome was explored. A strain with deletion of the pyocyanin synthesis gene phzM was constructed using homologous recombination-based seamless gene knockout technology. The growth and drug tolerance of the wild-type and mutant strains were compared.

Results Kyoto Encyclopedia of Genes and Genomes (KEGG) clustering analysis was performed on the website https://www.kegg.jp/. The results revealed a gene cluster composed of 9 genes (including PhzA, PhzB, PhzC, PhzD, PhzE, PhzF, PhzG, phzM and PhzS), which was completely consistent with the KEGG PATHWAY: Phenazine biosynthesis-M00835 Pyocyanine biosynthesis pathway. The key gene phzM for pyocyanin synthesis was knocked out via homologous recombination. After the exchange, monoclonal strains growing on M9-citrate medium were verified using primers phzM-AF and phzM-BR. The shuttle plasmid pK18mobSacB-phzM-AB was successfully transfected into the PAOII genotype strain. After gene exchange, monoclonal strains capable of growing on antibiotic-free plates were selected, and polymerase chain reaction (PCR) was performed to obtain strains with successful scarless knockout of the phzM gene. At 3 and 6 days of culture, the biofilm formation thickness of the PAOII genotype strain was greater than that of the PAOII-ΔphzM strain, with a statistically significant difference (P < 0.05).

Conclusion The biofilm synthesis capacity of the pyocyanin synthesis-deficient strain is reduced, and its resistance to cephalosporins (ceftazidime) is also decreased.