Transcriptional Regulation Of KP1₁373 Gene By Camp Receptor Protein （CRP） In Klebsiella Pneumoniae Ntuh-K2044

Objective: Klebsiella pneumoniae（K.pneumoniae）is a common and extremely important pathogenic bacteria among Gramnegative bacteria,which can cause a variety of diseases,such as pneumonia,urinary tract infections,blood infections,suppurative liver abscess and bacterial meningitis.cAMP receptor protein（CRP）is an important transcriptional regulator of bacteria.The function of KP1₁373 gene in Klebsiella pneumoniae is currently unclear.In the promoter region of the KP1₁373 gene,a binding consensus sequence for CRP protein was found,suggesting that CRP protein may affect the transcriptional regulation of the target gene.This article intends to explore and validate the possible regulatory effect of CRP protein on KP1₁373 gene by experiments.Methods: At first,the electrophoretic mobility shift assay（EMSA）was used to determine whether CRP protein could directly and specifically bind to the promoter region upstream of KP1₁373 gene.The DNA sequence of the promoter region of KP1₁373 gene was amplified and then incubated with purified CRP in vitro to observe whether there is a block band.Next,the LacZ fusion assay was performed to determine whether CRP regulated the target gene.The promoter sequence of KP1₁373 gene was amplified and cloned into the placZ15 plasmid to get the placZ15-p1373 recombinant plasmid.The recombinant plasmid was introduced into the lacZ-deficient Klebsiella pneumoniae CCW01 and the crp null mutant CCW01:Δcrp.The conclusion of the regulatory relationship of CRP protein on KP1₁373 gene was verified by comparing the β-galactosidase activity.Finally,the quantitative real-time PCR experiments（RT-qPCR）was carried out further to verify the transcriptional regulation of CRP on KP1₁373 gene.The total RNA of the Klebsiella pneumoniae wild strain NTUH-K2044（K2044）and the crp gene mutant K2044:Δcrp was extracted and reverse transcribed into cDNA to compare the relative expression of KP1₁373 gene in K2044 and K2044:Δcrp.Results: EMSA results indicated that CRP could directly bind to the promoter region of KP1₁373 gene.LacZ fusion assay showed that the β-galactosidase activity of CCW01:Δcrp strain was lower than that of CCW01 strain.RT-qPCR results showed that the relative expression of KP1₁373 in K2044:Δcrp was also lower than that in K2044.These results indicated that the transcriptional regulator CRP might positively affect the expression of KP1₁373 gene.Conclusions: The expression of KP1₁373 gene is affected by the transcriptional regulator CRP.The transcription of the KP1₁373 gene is positively regulated by CRP by directly binding to the motif of the promoter region.The above results provide a new recognition and basis for a clearer follow-up of the overall regulatory mechanism of CRP and the exploration of the function of the KP1₁373 gene.