Construction Of Klebsiella Pneumoniae GPKP Gene Kp05372 Deletion Strain And Analysis Of Its Biological Characteristics

Klebsiella Pneumoniae is an important opportunistic pathogen that can cause pneumonia and even death in musk deer.LysR transcriptional regulators in Klebsiella Pneumoniae are involved in the regulation of virulence,catabolism and drug resistance.The present study was aim to construct the deletion and replenishment of LysR transcriptional regulatory genes newly found in Klebsiella Pneumoniae GPKP from forest musk deer,and compare the biological characteristics differences among the three strains,including growth curve,drug sensitive test,biofilm,the resistance,the median lethal dose of bacteria,the colonization ability,and pathological morphology,as well as explore the fundamental function of the transcription regulatory factors.The main results are as follows:1.The construction of kp05372 gene deleted and complemented strains of Klebsiella Pneumoniae of musk deer:the upstream and downstream homologous arms of the new gene kp05372 were amplified,and the fused fragments were obtained by overlapping PCR.The recombinant suicide plasmids were successfully constructed by seamless clone connection using pLP12T plasmid as the vector.By conjugation transfer,chloramphenicol pressure forward screening and arabinose induction reverse screening,and after two homologous recombination,the missing strains GPKP-?kp05372 finally successfully was screened.The gene fragment kp05372 was cloned and linked with plasmid pBAD33 as vector,and the plasmid pBAD33-kp05372 was successfully constructed.By conjugation and translocation,arabinose induction and regulation of gene expression,the complement strain GPKP-?kp05372⁺was successfully screened.2.Biological characteristics analysis of Klebsiella pneumoniae of forest musk deer:there was no significant difference in the growth rate of the three strains of Klebsiella pneumoniae.All showed rapid growth after 2 h,reaching the maximum at 12 h,and decreased gradually after 18 h.The biofilm formation capacity of wild and supplemented strains was stronger than that of the deleted strains.The deleted strains were resistant to Cephalothiophene,compound Sinomin and Polymyxin B,and the inhibition zone diameter of gentamicin and ofloxacin was reduced by more than 5 mm,while the inhibition zone diameter of neomycin was increased by more than 5 mm.There was no significant change in osmotic pressure,temperature,salt content and hydrogen peroxide concentration.The acid tolerance of the deleted strains gradually decreased in the range of pH=2-7.The lethal dose of the wild strain,the deleted strain and the supplemented strain to mice was 6.3×10⁷ CFU/mL?1.1×10¹⁰ CFU/mL?4.88×10⁹ CFU/mL,respectively.After the content of the three strains in the liver decreased,the colonization remained stable.The content of the missing strains in the heart,spleen,kidney and intestines first decreased to 0,and the colonization ability was the weakest.All three strains could cause severe histopathological changes in liver and lung.In conclusion,new GPKP gene deletion strain GPKP-?kp05372 and replacement strain GPKP-?kp05372⁺were successfully constructed in this study.By comparing the differences in biological characteristics,it was found that the deletion of this gene resulted in decreased biofilm formation ability,changed drug resistance and decreased virulence of GPKP,and might also be involved in the regulation of the anti-acid mechanism of GPKP.The present study lays a foundation for further study on the regulatory mechanism of LysR type transcription regulator from GPKP gene kp05372.