Transcriptional Regulation Of KP1₀324 Grne By Camp Receptor Protein ?CRP? In Klebsiella Pneumoniae Ntuh-k2044

Purpose: Klebsiella pneumoniae?K.pneumoniae?is a common pathogen of nosocomial infections,which can cause urinary tract infections,pneumonia,sepsis,suppurative liver abscess and meningitis.cAMP receptor protein?CRP?is an important protein responsible for transcriptional regulation in bacteria.Bioinformatics analysis revealed a conserved sequence in the KP1₀324 promoter region that is likely to bind to CRP.KP1₀324 gene is homologous with yjcC that involved in biofilm formation.This study aimed to explore the regulation mechanism of CRP on KP1₀324 gene and the effect of KP1₀324 gene on biofilm formation.Methods: The labeled KP1₀324 fragment and purified CRP were incubated in vitro,and the electrophoretic mobility shift assay?EMSA?was used to analyze whether CRP binds to the KP1₀324 gene.Then,we used DNase I footprint to find the sequence of the binding sites of CRP and KP1₀324,and analyze its position relationship with the prediction binding sites.LacZ fusion assay and RT-qPCR were performed to study the transcriptional regulation of KP1₀324 by CRP.We constructed mutantstrains Kp-?0324 and complement strains Kpc-?0324.Crystal violet staining and scanning electron microscopy?SEM?were performed in K2044,Kp-?0324 and Kpc-?0324 to study the influence of KP1₀324 on biofilm formation.Results: The results of EMSA and DNase I footprint assay showed that CRP could specifically bind to KP1₀324,and the binding sites were391 to 350 upstream of the KP1₀324 start codon ATG.LacZ fusion assay and RT-qPCR showed CRP negatively regulated the KP1₀324.We found the deletion of KP1₀324 gene weakened the ability of the biofilm formation.Conclusions: CRP negatively regulate KP1₀324 gene involved in biofilm formation.These results provide insight into the CRP regulatory networks of K.pneumoniae.