Abstract: 【Objective】The objective of this study is to investigate the molecular mechanism of H-NS regulating conjugation of the IncFⅡ plasmid from a clinically isolated multi-drug resistant Escherichia coli from chicken. The aim is to provide a theoretical basis for controlling the rapid spread of IncFⅡ plasmid mediated multidrug resistance genes.【Method】The growth curves of Escherichia coli ATCC25922 and four recombinant strains (F25922, pBAD25922, FΔhns and FΔhns/phns) were determined to compare the influence of hns on different strains. Conjugation experiments were conducted with F25922, FΔhns and FΔhns/phns as donors and Escherichia coli J53 as recipient, then the conjugation frequency was calculated. The mRNA expression levels of IncFⅡ plasmid conjugation transfer related genes(traM, traJ and traY) in each recombinant strains(F25922, FΔhns and FΔhns/phns) were detected by RT-qPCR. The LacZ reporter strains F25922/P_M(P_J/P_Y), FΔhns/P_M(P_J/P_Y) and FΔhns/phns/P_M(P_J/P_Y) were constructed to determine the β-galactosidase activity of three promoters of tra genes(traM, traJ and traY). The H-NS protein was purified by Ni-NTA resin affinity chromatography. The DNA sequences of three promoters of tra genes were amplified by PCR. The mechanism of H-NS regulating IncFⅡ plasmid transmission was identified by EMSA. And the binding sites of H-NS to different promoters were predicted and further verified by ESMA.【Results】The growth of recombinant strains F25922 and pBAD25922 were not significantly different from that of Escherichia coli ATCC25922, the growth rate of deleted recombinant strain FΔhns and complemented strain FΔhns/phns were significantly lower than that of the control strain F25922. The results showed that the absence of hns could make the adaptability of strains worse, but did not affect the survival of the strains. The results of the conjugation test showed that the conjugation frequency of IncFⅡ plasmid in FΔhns was 1279.33 times higher than that of the control strain F25922(P<0.001), and the FΔhns/phns conjugation frequency of the supplementary strain was significantly lower than that of FΔhns, although it did not completely recover to the level of the control strain F25922. Similarly, the mRNA expression levels of these tra genes(traM, traJ and traY) were significantly higher in the deletion mutant FΔhns. The mRNA expression level of traJ was the highest in FΔhns, which was 1510.14 times that of F25922, followed by traY and traM, which were 448.14 times and 81.54 times that of F25922, respectively. Compared to the deletion strain FΔhns, expression levels of the tra genes(traM, traJ and traY) in the complemented strain FΔhns/phns were significantly decreased. The β-Galactosidase activities of promoters P_M, P_J and P_Y in the reporter strains FΔhns/P_M(P_J/P_Y) were 5.66, 10.45 and 21.91, respectively, which were significantly higher than that of the corresponding promoters of F25922/P_M(P_J/P_Y) (P<0.001). The activities of promoters P_M, P_J and P_Y in the complement reporter strains FΔhns/phns/P_M(P_J/P_Y) were significantly lower than that of FΔhns/P_M(P_J/P_Y), and there was no significant difference with the control strain F25922/P_M(P_J/P_Y). EMSA results showed that H-NS protein could block the DNA migration of three promoters of tra genes, indicating that H-NS could directly bind to the three promoters. By predicting the binding sites and further verified by EMSA, it was confirmed that H-NS protein could bind directly to the AT enrichment region of the promoters of the three genes(traM, traJ and traY).【Conclusion】H-NS protein could bind directly to the AT enrichment region of the promoter region of IncFⅡ plasmid conjugation transfer related genes(traM, traJ and traY), and negatively regulate the conjugation transfer of IncFⅡ plasmid by inhibiting the activity of promoter.

Key words: H-NS, tra gene, IncFⅡ, Plasmid mating, negative regulation, E. coli from chicken