Functions of mutants of diguanylate cyclase SiaD from Pseudomonas aeruginosa

Abstract:

[Objective] Diguanylate cyclase SiaD regulates the biofilm formation of Pseudomonas aeruginosa.Our previous study about the effect of siaD overexpression on biofilm has revealed that the biofilm yield of a complementary strain is significantly higher than that of the strain overexpressing the wild-type siaD gene.This study aims to explore the reasons for the increase in biofilm production and to study other phenotypes of this strain.[Methods] The mutation sites of siaD were identified by sequencing.The qualitative and quantitative biofilm experiments were carried out to analyze the phenotype of the strain with point mutation.Western blotting was employed to determine the protein level of SiaD^R119M,and GST-pull down assay to measure the binding ability of SiaC to SiaD^R119M in vitro.The fusion expression vector was constructed for the point mutation gene siaD^R119M,and the protein was expressed and purified.The enzyme activity of SiaD^R119M was detected by high performance liquid chromatography (HPLC).Further,we studied the motility of the strain to reveal the relationship between c-di-GMP and bacterial motility.[Results] The sequencing comparison showed that the 119th amino acid was mutated from arginine to methionine (R119M).Compared with that of the wild-type siaD complementary strain,the biofilm yield of siaD^R119A increased,while the biofilm yield of siaD^R119A was lower than that of siaD^R119M;the biofilm yield of siaD^R201A significantly increased and was higher than that of siaD^{R119M R201A}.Western blotting showed no difference in the expression level between SiaD^R119M and wild-type SiaD,and the GST-pull down assay indicated there was a specific interaction between SiaC and SiaD^R119M.The HPLC results showed that the activity of SiaD^R119M decreased.Compared with wild-type siaD complementary strain,siaD^R119M showed weakened motility,and siaD^R201A and siaD^{R119M R201A} had no significant difference in motility.[Conclusion] The mutation of R119M in SiaD increased the biofilm yield,decreased the enzyme activity,and reduced the bacterial motility.This mutation may affect the interaction between SiaD and downstream effector to enhance the signal transduction of downstream effector.The underlying mechanism remains to be explored.

Key words:siaD^R119M mutant;biofilm;c-di-GMP;motility