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2022, Vol. 26 ›› Issue (17): 2738-2743Construction of pgaABCD gene deletion and complementation strains and properties of biofilm formation ability for Escherichia coliGong Haiyan1, Cheng Qian2, Zhao Zhilong1, Shi Yang3
Abstract: BACKGROUND: Escherichia coli can cause difficult refractory infections, which seriously threaten human health, due to the formation of “biofilms.” The pgaABCD opero is one of the important regulatory genes for biofilm formation. OBJECTIVE: To construct gene deletion strain MG1655/Δpga of Escherichia coli, construct the complementation strains of Δpga/pgaA, Δpga/pgaB, Δpga/pgaC, Δpga/pgaD by complementing pgaA, pgaB, pgaC and pgaD, respectively, observe the biofilm formation ability, and exopolysaccharide and morphological characteristics of deletion and complementation strains, and analyze the effects of gene pga on the regulation and characteristics of biofilm. METHODS: Based on the Lambda Red recombination system, helper plasmid pKD46 was transferred into competent cells MG1655 to obtain MG1655/pKD46 monoclones. Targeting fragment of pgaABCD gene was transformed to competent cells MG1655/pKD46 to construct gene deleted mutant of MG1655/Δpga. Using plasmid pBR322 as vector, pBR322-pgaA, pBR322-pgaB, pBR322-pgaC, and pBR322-pgaD were constructed and transformed into MG1655/Δpga to obtain the corresponding complementation strains. Congo red-broth agar plate, crystal violet semi-quantitative method, and transmissionelectronmicroscopy were utilized to detect property changes of biofilm formation capacity of construction strains. At the same time, extracellular polysaccharide was detected using phenol-sulfuric acid method. RESULTS AND CONCLUSION: The biofilm formation capacity and extracellular polysaccharide of deletion strains were significantly decreased (P < 0.05). Some cells were damaged and collapsed. The section showed vacuolardegeneration in bacteria. To conclude, deletion of gene pgaABCD can affect the biofilm characteristics for Escherichia coli, and the genes pgaA, pgaB, pgaC and pgaD, but not the only regulatory gene, are involved in the biofilm formation. Key words:Escherichia coli, pgaABCD, biofilm, extracellular polysaccharide, deletion strain, complementation strain |