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

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"

[1] LIU Y Y, WANG Y, WALSH T R, YI L X, ZHANG R, SPENCER J, DOI Y, TIAN G, DONG B, HUANG X, YU L F, GU D, REN H, CHEN X, LV L, HE D, ZHOU H, LIANG Z, LIU J H, SHEN J. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet Infectious Diseases, 2016, 16(2): 161-168. doi: 10.1016/s1473-3099(15)00424-7.
doi: 10.1016/s1473-3099(15)00424-7
[2] WOODFORD N, CARATTOLI A, KARISIK E, UNDERWOOD A, ELLINGTON M J, LIVERMORE D M. Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25: H4-ST131 clone. Antimicrobial Agents and Chemotherapy, 2009, 53(10): 4472-4482. doi: 10.1128/aac.00688-09.
doi: 10.1128/aac.00688-09
[3] JIAN H, XU G, GAI Y, XU J, XIAO X. The histone-like nucleoid structuring protein (H-NS) is a negative regulator of the lateral flagellar system in the deep-sea bacterium Shewanella piezotolerans WP3. Applied and Environmental Microbiology, 2016, 82(8): 2388-2398. doi: 10.1128/aem.00297-16.
doi: 10.1128/aem.00297-16
[4] VAN DER MAAREL J R, GUTTULA D, ARLUISON V, EGELHAAF S U, GRILLO I, FORSYTH V T. Structure of the H-NS-DNA nucleoprotein complex. Soft Matter, 2016, 12(15): 3636-3642. doi: 10.1039/c5sm03076e.
doi: 10.1039/c5sm03076e
[5] 徐亚珂, 龙金照, 段广才, 梁文娟, 刘慧莹, 陈帅印, 郗园林. CTX-M-15型大肠埃希菌的分子分布特征及质粒传播规律研究. 中国病原生物学杂志, 2017(11): 1048-1051.
XU Y K, LONG J Z, DUAN G C, LIANG W J, LIU H Y, CHEN S Y, XI Y L. The molecular distribution characteristics of and plasmid horizontal transfer in CTX-M-15-positive Escherichia coli. Journal of Pathogen Biology, 2017(11): 1048-1051. (in Chinese)
[6] PAN Y S, ZONG Z Y, YUAN L, DU X D, HUANG H, ZHONG X H, HU G Z. Complete sequence of pEC012, a multidrug-resistant IncI1 ST71 plasmid carrying blaCTX-M-65, rmtB, fosA3, floR, and oqxAB in an avian Escherichia coli ST117 strain. Frontiers in Microbiology, 2016, 7: 1117.
[7] DIACONU E L, CARFORA V, ALBA P, DI MATTEO P, STRAVINO F, BUCCELLA C, DELL'AIRA E, ONORATI R, SORBARA L, BATTISTI A, FRANCO A. Novel IncFⅡ plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy, 2020, 75(12): 3475-3479.
doi: 10.1093/jac/dkaa374
[8] XU L, WANG P, CHENG J, QIN S, XIE W. Characterization of a novel blaNDM-5-harboring IncFII plasmid and an mcr-1-bearing IncI2 plasmid in a single Escherichia coli ST167 clinical isolate. Infection and Drug Resistance, 2019, 12: 511-519. doi: 10.2147/idr.s192998.
doi: 10.2147/idr.s192998
[9] DENG Y, ZENG Z, CHEN S, HE L, LIU Y, WU C, CHEN Z, YAO Q, HOU J, YANG T, LIU J H. Dissemination of IncFⅡ plasmids carrying rmtB and qepA in Escherichia coli from pigs, farm workers and the environment. Clinical Microbiology and Infection, 2011, 17(11): 1740-1745.
doi: 10.1111/j.1469-0691.2011.03472.x
[10] PICKER M A, WING H J. H-NS, its family members and their regulation of virulence genes in Shigella species. Genes (Basel), 2016, 7(12): 112.
doi: 10.3390/genes7120112
[11] HÜTTENER M, PAYTUBI S, JUÁREZ A. Success in incorporating horizontally transferred genes: the H-NS protein. Trends in Microbiology, 2015, 23(2): 67-69. doi: 10.1016/j.tim.2014.12.009.
doi: 10.1016/j.tim.2014.12.009
[12] TENDENG C, BERTIN P N. H-NS in Gram-negative bacteria: a family of multifaceted proteins. Trends in Microbiology, 2003, 11(11): 511-518. doi: 10.1016/j.tim.2003.09.005.
doi: 10.1016/j.tim.2003.09.005
[13] RANGARAJAN A A, SCHNETZ K. Interference of transcription across H-NS binding sites and repression by H-NS. Molecular Microbiology, 2018, 108(3): 226-239. doi: 10.1111/mmi.13926.
doi: 10.1111/mmi.13926
[14] JACQUET M, CUKIER-KAHN R, PLA J, GROS F. A thermostable protein factor acting on in vitro DNA transcription. Biochemical and Biophysical Research Communications, 1971, 45(6): 1597-1607.
doi: 10.1016/0006-291X(71)90204-X
[15] DAME R T, NOOM M C, WUITE G J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature, 2006, 444(7117): 387-390.
doi: 10.1038/nature05283
[16] DAME R T, WYMAN C, GOOSEN N. H-NS mediated compaction of DNA visualised by atomic force microscopy. Nucleic Acids Research, 2000, 28(18): 3504-10.
doi: 10.1093/nar/28.18.3504
[17] HELGESEN E, FOSSUM-RAUNEHAUG S, SKARSTAD K. Lack of the H-NS protein results in extended and aberrantly positioned DNA during chromosome replication and segregation in Escherichia coli. Journal of Bacteriology, 2016, 198(8): 1305-1316. doi: 10.1128/jb.00919-15.
doi: 10.1128/jb.00919-15
[18] ALI S S, SOO J, RAO C, LEUNG A S, NGAI D H, ENSMINGER A W, NAVARRE W W. Silencing by H-NS potentiated the evolution of Salmonella. PloS Pathogens, 2014, 10(11): e1004500.
doi: 10.1371/journal.ppat.1004500
[19] SERNA A, ESPINOSA E, CAMACHO E M, CASADESÚS J. Regulation of bacterial conjugation in microaerobiosis by host- encoded functions ArcAB and sdhABCD. Genetics, 2010, 184(4): 947-958. doi: 10.1534/genetics.109.109918.
doi: 10.1534/genetics.109.109918
[20] LU J, FROST L S. Mutations in the C-terminal region of TraM provide evidence for in vivo TraM-TraD interactions during F-plasmid conjugation. Journal of Bacteriology, 2005, 187(14): 4767-4773.
doi: 10.1128/JB.187.14.4767-4773.2005
[21] WILL W R, LU J, FROST L S. The role of H-NS in silencing F transfer gene expression during entry into stationary phase. Molecular Microbiology, 2004, 54(3): 769-782. doi: 10.1111/j.1365-2958.2004.04303.x.
doi: 10.1111/j.1365-2958.2004.04303.x.
[22] WILL W R, FROST L S. Characterization of the opposing roles of H-NS and TraJ in transcriptional regulation of the F-plasmid tra operon. Journal of Bacteriology, 2006, 188(2): 507-514. doi: 10.1128/jb.188.2.507-514.2006.
doi: 10.1128/jb.188.2.507-514.2006
[23] PAN Y S, YUAN L, ZONG Z Y, LIU J H, WANG L F, HU G Z. A multidrug-resistance region containing blaCTX-M-65, fosA3 and rmtB on conjugative IncFⅡ plasmids in Escherichia coli ST117 isolates from chicken. Journal of Medical Microbiology, 2014, 63(Pt 3): 485-488.
doi: 10.1099/jmm.0.070664-0
[24] 胡慧慧, 孙亚伟, 李文娅, 邝启红, 孙华润, 吴华, 胡功政, 苑丽. 大肠杆菌cpxRhns双基因缺失株的构建. 中国兽医学报, 2020(1): 116-121.
HU H H, SUN Y W, LI W Y, KUANG Q H, SUN H R, WU H, HU G Z, YUAN L. Construction of cpxR and hns double gene deletion strain of Escherichia coli. Chinese Journal of Veterinary Science, 2020(1): 116-121. (in Chinese)
[25] HUANG H, SUN Y, YUAN L, PAN Y, GAO Y, MA C, HU G. Regulation of the two-component regulator CpxR on aminoglycosides and β-lactams resistance in Salmonella enterica serovar Typhimurium. Frontiers in Microbiology, 2016, 7: 604. doi: 10.3389/fmicb.2016.00604.
doi: 10.3389/fmicb.2016.00604
[26] DE LA CRUZ M A, RUIZ-TAGLE A, ARES M A, PACHECO S, YÁÑEZ J A, CEDILLO L, TORRES J, GIRÓN J A. The expression of Longus type 4 Pilus of enterotoxigenic Escherichia coli is regulated by LngR and LngS and by H-NS, CpxR and CRP global regulators. Environmental Microbiology, 2017, 19(5): 1761-1775. doi: 10.1111/1462-2920.13644.
doi: 10.1111/1462-2920.13644
[27] HUANG F, FITCHETT N, RAZO-GUTIERREZ C, LE C, MARTINEZ J, RA G, LOPEZ C, GONZALEZ L J, SIEIRA R, VILA A J, BONOMO R A, RAMIREZ M S. The H-NS regulator plays a role in the stress induced by carbapenemase expression in Acinetobacter baumannii. mSphere, 2020, 5(4): e00793-20.
[28] WILL W R, BALE D H, REID P J, LIBBY S J, FANG F C. Evolutionary expansion of a regulatory network by counter- silencing. Nature Communications, 2014, 5: 5270. doi: 10.1038/ncomms6270.
doi: 10.1038/ncomms6270
[29] LU J, PENG Y, WAN S, FROST L S, RAIVIO T, GLOVER JNM. Cooperative function of TraJ and ArcA in regulating the F plasmid tra operon. Journal of Bacteriology, 2018, 201(1): e00448-18.
[30] WAN B, ZHANG Q, TAO J, ZHOU A, YAO Y F, NI J. Global transcriptional regulation by H-NS and its biological influence on the virulence of Enterohemorrhagic Escherichia coli. Gene, 2016, 588(2): 115-123. doi: 10.1016/j.gene.2016.05.007.
doi: 10.1016/j.gene.2016.05.007
[31] ARES M A, FERNÁNDEZ-VÁZQUEZ J L, ROSALES-REYES R, JARILLO-QUIJADA M D, VON BARGEN K, TORRES J, GONZÁLEZ-Y-MERCHAND J A, ALCÁNTAR-CURIEL M D, DE LA CRUZ M A. H-NS nucleoid protein controls virulence features of Klebsiella pneumoniae by regulating the expression of type 3 pili and the capsule polysaccharide. Frontiers in Cellular and Infection Microbiology, 2016, 6: 13. doi: 10.3389/fcimb.2016.00013.
doi: 10.3389/fcimb.2016.00013
[32] BISCHOF K, SCHIFFER D, TRUNK S, HÖFLER T, HOPFER A, RECHBERGER G, KORAIMANN G. Regulation of R1 plasmid transfer by H-NS, ArcA, TraJ, and DNA sequence elements. Frontiers in Microbiology, 2020, 11: 1254. doi: 10.3389/fmicb.2020.01254.
doi: 10.3389/fmicb.2020.01254
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