Cloning, expression, purification, and biochemical characterization of CpxR protein from pectobacterium carotovorum.
CpxR
circular dichroism
gene expression
pectobacterium carotovorum
protein purification
Journal
Biotechnology and applied biochemistry
ISSN: 1470-8744
Titre abrégé: Biotechnol Appl Biochem
Pays: United States
ID NLM: 8609465
Informations de publication
Date de publication:
Jun 2022
Jun 2022
Historique:
received:
17
12
2020
accepted:
26
03
2021
pubmed:
4
4
2021
medline:
7
7
2022
entrez:
3
4
2021
Statut:
ppublish
Résumé
The cpxR gene, encoding a new cytoplasmic response regulator, which effects virulence, biofilm formation, chemotaxis, resistance to antimicrobials, and controls soft rot, was amplified by the polymerase chain reaction, cloned into the prokaryotic expression vector pET-15b, and expressed through the induction of isopropyl-β-d-thiogalactoside in Escherichia coli BL21 (DE3). Then, highly purified and stable CpxR protein was produced by nickel affinity chromatography and fast protein liquid chromatography, digested by thrombin and identified by Western blotting. Furthermore, the structure of the CpxR protein was estimated by circular dichroism spectroscopy and SWISS-MODEL. The CpxR protein was a functional part in signal transduction and bacterial resistance for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The resear ch of the protein stability indicated the CpxR protein had excellent thermal stability and was suitable for crystallization. Then the small crystals of CpxR protein were found in the crystallizing tank. The latest 34 cpxR sequences from the public database were selected and analyzed by molecular clustering and multisequence alignment. These cpxR sequences were roughly divided into four categories. These results laid an important foundation for the further structural study of the CpxR protein.
Substances chimiques
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
898-905Informations de copyright
© 2021 International Union of Biochemistry and Molecular Biology, Inc.
Références
Nevesinjac AZ, Raivio TL. The Cpx envelope stress response affects expression of the type IV bundle-forming pili of enteropathogenic Escherichia coli. J.Bacteriol. 2005;187(2):672-86.
Miguel AD, Deyanira P, Irene JP, Marcos F, Edmundo C, Victor HB. The two-component system CpxRA represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD. Front Microbiol. 2015;6: 807.
Wolfe AJ, Parikh N, Lima BP, Cementitis B. Signal integration by the two-component signal transduction response regulator CpxR. J. Bacteriol. 2008;190(7):2314-22.
Gal-Mor O, Segal G Identification of CpxR as a positive regulator of icm and dot virulence genes of legionella pneumophila. J. Bacteriol. 2003;185(16):4908-19.
Raivio TL. Everything old is new again: an update on current research on the Cpx envelope stress response. Biochim Biophys Acta. 2014;1843(8):1529-41.
Altman E, Segal G. The response regulator CpxR directly regulates expression of several legionella pneumophila icm/dot components as well as new translocated substrates. J Bacteriol. 2008;190(6):1985-96.
Wulf P.De., McGuire AM, Liu X, Lin EC. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli. J Biol Chem. 2002;277(29):26652-61.
Gangaiah D, Spinola SM, Zhang X, Fortney KR, Baker B, Liu Y, et al. Activation of CpxRA in Haemophilus ducreyi primarily inhibits the expression of its targets, including major virulence determinants. J Bacteriol. 2002;195(15):3486-502.
Weatherspoon-Griffin N, Yang D, Kong W, Hua Z, Shi Y. The CpxR/CpxA two-component regulatory system up-regulates the multidrug resistance cascade to facilitate Escherichia coli resistance to a model antimicrobial peptide. J Biol Chem. 2014;289(47):32571-82.
Zhou X, Keller R, Volkmer R. Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein. J Biol Chem. 2011;286(11):9805-14.
Zhou X, He X, Xu L. Cloning and expression of cpxA gene from Pectobacterium carotovorum subsp. Carotovorum. J Chem Pharma Res. 2014;6(6):396-400.
Hunke S, Keller R, Muller VS Signal integration by the Cpx-envelope stress system. FEMS Microbiol Lett. 2012;326(1):12-22.
He XL, Zhou XH, Yang Z, Xu L, Yu YX, Jia LL, et al. Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109. Protein Expres Purif. 2015;114:77-81.
Yang Z, Zhou X, Song J, Fang X, Xiong X. Construction and expression of CpxP protein from plant pathogenic bacteria. In: International Conference on Biomedical Engineering and Biotechnology. IEEE: 2012. pp. 119-22.
He XL, Ren YW, Meng WL, Yu XR, Zhou XH. Knocking out analysis of the CpxP gene using Crispr/Cas9 in Escherichia coli MG1655. AMB Exp. 2020;10: 172.
Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 2012;13(6):614-29.
Lee DH, Lim JA, Lee J, Roh E, Jung K, Choi M, et al. Characterization of genes required for the pathogenicity of Pectobacterium carotovorum subsp. carotovorum PCC21 in Chinese cabbage. Microbiology. 2013;159(7):1487-96.
Raivio TL. Envelope stress responses and Gram-negative bacterial pathogenesis. Mol Microbiol. 2005;56:1119-28.
Ma Q, Wood TK OmpA influences Escherichia coli biofilm formation by repressing cellulose production through the CpxRA two-component system. Environ Microbiol. 2009;11:2735-46
De Wulf P, Kwon O, Lin EC The CpxRA signal transduction system of Escherichia coli: growth-related autoactivation and control of unanticipated target operons. J Bacteriol. 1999;181:6772-8.
Weatherspoon-Griffin N, Zhao G, Kong W, Kong Y, Morigen, A-P. H., et al. The CpxR/CpxA two-component system up-regulates two Tat-dependent peptidoglycan amidases to confer bacterial resistance to antimicrobial peptide. J Biol Chem. 2011;286:5529-39.
Hirakawa H, Nishino K, Hirata T, Yamaguchi A Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J Bacteriol. 2003;185:1851-6.
Nishino K, Yamasaki S, Hayashi-Nishino M, Yamaguchi A Effect of NlpE overproduction on multidrug resistance in Escherichia coli. Antimicrob Agents Chemother. 2010;54: 2239-43.
Mahoney TF, Silhavy TJ. The Cpx stress response confers resistance to some, but not all, bactericidal antibiotics. J Bacteriol. 2013;195:1869-74.