The Investigate Of Regulation Mechanism Of Pseudomonas Aeruginosa LysR Regulatory Protein Pa0191 In Response To Iron Regulation

Pseudomonas aeruginosa is an important pathogen that causes hospital-acquired infections and it can cause severe acute and chronic infections.Iron is one of the important necessary elements for the growth of P.aeruginosa,which is related to its virulence.Pyoverdine（PVD）is an important virulence factor secreted by P.aeruginosa,which is used to efficiently uptake and chelate iron from the environment.PVD is related to the biosynthesis of biofilm and the pathogenicity to the host.RNA-seq analysis showed that in P.aeruginosa PAO1,the deletion of cys B resulted in a significant increase in the expression of pa0191-pa0194 gene cluster at the transcriptional level,and the deletion of pa0191 resulted in a significant increase in the expression of genes encoding multiple PVD-related proteins at the transcriptional level.In this thesis,we focused on the function and regulation of pa0191-pa0194 gene cluster.Bioinformatics analysis showed that PA0191 is a Lys R family transcriptional factor.PA0192 is a Ton B-dependent outer membrane receptor protein that may be related to the transportation of small moleculars.PA0193 and PA0194 are Tau D/Tfd A family dioxygenases with the function of chelating Fe²⁺.Through the detection of gene transcription level,it was found that PA0191-0194 is down regulated by the transcriptional regulator Cys B,and electrophoretic mobility shift assay also proved that Cys B can bind to the promoter-pa0192.The expressions of multiple protein-coding genes related to PVD synthesis were down regulated by PA0191verified by Real-time Quantitativepolymerase chain reaction The results of EMSA and q RT-PCR were illustrated together,PA0191 is able to bind to the promoter-pa0192 and up regulate the expressions of pa0192-pa0194 directly.Under iron-repletion condition of high-iron,the transcription level expression of pa0191-pa0194 is decreased.Less PA0191 bound to the promoter-pa0192,and,the expressions of pa0192-pa0194 decreased under iron-repletion condition.The expressions of pa0191-pa0194 and the regulation process of PA0191 on pa0192-pa0194 were affected by iron concentration.The single deletion of pa0191-pa0194 gene did not affect the growth,the metabolism and iron uptake in P.aeruginosa PAO1 by the growth curve detection experiment,biofilm experiment and intracellular iron concentration determination experiment.PA0191-PA0194 is not the only way for P.aeruginosa PAO1 to metabolize iron.In summary,we revealed that the growth metabolism and virulence expression of P.aeruginosa PA0191 was regulated by iron,and it was also found that PA0191-PA0194 gene cluster was not the main factor involving iron uptake by P.aeruginosa.In order to maintain normal growth metabolism,P.aeruginosa might uptake and utilize iron through other compensation mechanisms.The results of this study provides important ideas for understanding the environmental adaptability of P.aeruginosa and the uptake of iron ions,which provides a theoretical basis for further development of more targeted bacterial therapy drugs.