Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (18): 3675-3684.doi: 10.3864/j.issn.0578-1752.2022.18.016

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Molecular Mechanism of Regulation by H-NS on IncFⅡ Plasmid Transmission of Multi-drug Resistant Chicken Escherichia coli

YaTing JIA(),HuiHui HU,YaJun ZHAI,Bing ZHAO,Kun HE,YuShan PAN,GongZheng HU,Li YUAN   

  1. College of Animal Medicine, Henan Agricultural University, Zhengzhou 450046
  • Received:2021-08-02 Accepted:2021-10-27 Online:2022-09-16 Published:2022-09-22
  • Contact: YUAN Li E-mail:1218460153@qq.com

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Abstract:

【Objective】The objective of this study was 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, so as 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. The 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/PM(PJ/PY), FΔhns/PM(PJ/PY) and FΔhns/phns/PM(PJ/PY) 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.【Result】The growth of recombinant strains F25922 and pBAD25922 were not significantly different from that of Escherichia coli ATCC25922, while 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 1 279.33 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 1 510.14 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 PM, PJ and PY in the reporter strains FΔhns/PM (PJ/PY) were 5.66, 10.45 and 21.91, respectively, which were significantly higher than that of the corresponding promoters of F25922/PM (PJ/PY) (P<0.001). The activities of promoters PM, PJ and PY in the complement reporter strains FΔhns/phns/PM (PJ/PY) were significantly lower than that of FΔhns/PM (PJ/PY), and there was no significant difference with the control strain F25922/PM (PJ/PY). 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

Table 1

Sequences of primers used in this study"

引物 Primers 序列(5′→3′) Squence (5′→3′) 产物 Product size/bp 参考文献 Reference
RT-qPCR所用到的引物及序列(Sequences of primers used in RT-qPCR)
traY-F
traY-R		 TATAAGCAGAACAGTCTCGGTGTA
GACGGATCTGGACTTCTATCGT		 107
traJ-F
traJ-R		 GGAAAATCCTTTATGCCAATG
AGAGCCATGTCCAAGAGCCT		 146
traM-F
traM-R		 TTGAAAAGCGTCGTCAGGAG
CGCAGACTCTTTACGCTCCA		 122
16SrRNA-F
16SrRNA-R		 GTGTAGCGGTGAAATGCG
CGTTTACGGCGTGGACTA		 136 [25]
构建融合报告质粒所用到的引物及序列( Sequences of primers used in fusion report plasmids were constructed)
PM-F
PM-R		 CCGGAATTC TAGCTTATGTTTTAAATA
CGCGGATCC AAGACAGTACCTTTTATA		 276
PJ-F
PJ-R		 CCGGAATTC AGGTATTGAGTCTCTCAG
CCGGGATCCAGGAACCTCCGTTAAGGA		 320
PY-F
PY-R		 CCGGAATTCTCAGAGTGGAAGTAATAC
CCGGGATCCTCAATTCACCTCTGTTTC		 379
pKP302-F
pKP302-R		 TGATTTTGTGCCGAGCTGCCGGTC
GGCGAAAGGGGGATGTGCTGC		 305/548/592/651
EMSA所用到的引物及序列 (Primers and sequences used in EMSA)
PM483-F
PM483-R		 AAAGGCTCAACAGGTTGGTG
GAAGCAGTTCCTGAAAGGCT		 483
PJ407-F
PJ407-R		 CCGGAAACCCGAAGTTTGAA
TTGTTACGGACACAAACCGG		 407
PY377-F
PY377-R		 CAGAGCATCTGAGGATCGAA
TGAGACGGATCTGGACTTCT		 377
PM25-F
PM25-R		 TTGTTTCAGAATATAAAAGGTACTG
CAGTACCTTTTATATTCTGAAACAA		 25
PJ25-F
PJ25-R		 ATTGAAACTGAAAATCGCCGATGCA
TGCATCGGCGATTTTCAGTTTCAAT		 25
PY20-F
PY20-R		 GTTAAGTAAATGTTAAATAA
TTATTTAACATTTACTTAAC		 20

Fig. 1

Growth curves of hns deleted strains25922: Escherichia coli ATCC25922; F25922, FΔhns and FΔhns/phns were recombinant strains carrying pEC011(IncFⅡ) plasmid, which were transferred into Escherichia coli ATCC25922, Δhns and Δhns/phns, respectively. pBAD25922 was a recombinant strain which pBAD plasmid was electrotransferred into Escherichia coli ATCC25922"

Fig. 2

Conjugation transfer frequencies for IncFⅡ plasmid pEC011 F25922, FΔhns and FΔhns/phns were recombinant strains carrying pEC011(IncFⅡ) plasmid, which were transferred into Escherichia coli ATCC25922, Δhns and Δhns/phns, respectively. *** means there was a statistically significant difference (P<0.001), P represents the difference of transfer frequencies of IncFⅡ plasmid between FΔhns and F25922, FΔhns and FΔhns/phns, respectively"

Fig. 3

The expression levels of 3 selected transfer genes involved in conjugation F25922, FΔhns and FΔhns/phns were recombinant strains carrying pEC011(IncFⅡ) plasmid, which were transferred into Escherichia coli ATCC25922, Δhns and Δhns/phns, respectively. traM, traJ and traY were pEC011(IncFⅡ) conjugation related genes. *** means there was a significant difference(P<0.001), P represents the mRNA expression levels of different tra genes(traM、traJ and traY) in FΔhns and F25922"

Fig. 4

β-galactosidase activity of difference promoters of reporter strains F25922, FΔhns and FΔhns/phns were recombinant strains carrying pEC011(IncFⅡ) plasmid, which were transferred into Escherichia coli ATCC25922, Δhns and Δhns/phns, respectively. PY, PJ and PM were promoters of pEC011(IncFⅡ) plasmid conjugation related genes traY, traJ and traM. *** means there was a significant difference(P<0.001) of β-galactosidase activity of the same promoters between FΔhns and F25922. means there was a significant difference(P<0.001) of β-galactosidase activity between PY and PJ or PJ and PM in FΔhns"

Fig. 5

H-NS protein binding to the promoters of tra gene PM, PJ and PJ were promoters of pEC011(IncFⅡ) plasmid conjugation related genes traM, traJ and traY , respectively. The length of them were: PM: 483 bp; PJ: 403 bp; PY: 377 bp"

Fig. 6

The binding sites of H-NS protein and the promoters of tra genes PM25, PJ25 and PY25were promoters of pEC011(IncFⅡ) plasmid conjugation related genes traM, traJ and traY , respectively. The length of them were: PM25: 25 bp; PJ25: 25 bp; PY20: 20 bp"

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