Regulation of Efflux Pump MdrL by LadR in Listeria monocytogenes

doi:10.13560/j.cnki.biotech.bull.1985.2018-0430

Abstract

Abstract: The aim of this study is to investigate the role of LadR in the regulation of efflux pump MdrL in Listeria monocytogenes. The mutant strain ΔladR was constructed from the wild type strain EGD-e by homologous recombination. Thetranscriptional levels of mdrL in the wild type strain EGD-e and the mutant strain ΔladR were tested by quantitative reverse transcriptase PCR. The expression strain BL21（DE3）-ladR was constructed,and then the recombination protein His₆-LadR was purified. Electrophoretic mobility shift assay（EMSA）was employed to assess the binding activity of LadR to the mdrL promoter. As results,the mutant strain ΔladR was constructed successfully. Compared to the wild type strain EGD-e,the transcriptional level of mdrL in ΔladR showed about 51-fold up-regulation. The expression strain BL21（DE3）-ladR was also constructed successfully,and the concentration of the purified recombinant protein His₆-LadR was 1 mg/mL. Results of EMSA showed that His₆-LadR protein was able to bind to the promoter of gene mdrL specifically. Conclusively,the regulator LadR negatively regulates the efflux pump MdrL by specifically binding to the promoter of mdrL.

Key words: Listeria monocytogenes, LadR, efflux pump, regulation

XU Ya-meng, JIANG Xiao-bing, YU Tao. Regulation of Efflux Pump MdrL by LadR in Listeria monocytogenes[J]. Biotechnology Bulletin, 2018, 34(12): 166-171.

References

[1] Arslan F, Meynet E, Sunbul M, et al.The clinical features, diagnosis, treatment, and prognosis of neuroinvasive listeriosis:a multinational study[J]. European Journal of Clinical Microbiology & Infectious Diseases, 2015, 34(6):1213-1221.
[2] Ryan E, Gahan C, Hill C.A significant role for Sigma B in the detergent stress response of Listeria monocytogenes[J]. Letters in Applied Microbiology, 2008, 46(2):148-154.
[3] Filipello V, Gallina S, Amato E, et al.Diversity and persistence of Listeria monocytogenes, within the Gorgonzola PDO production chain and comparison with clinical isolates from the same area[J]. International Journal of Food Microbiology, 2017, 245:73-78.
[4] Jiang X, Yu T, Liang Y, et al.Efflux pump-mediated benzalkonium chloride resistance in Listeria monocytogenes isolated from retail food[J]. International Journal of Food Microbiology, 2016, 217:141-145.
[5] Martínez-Suárez JV, Ortiz S, López-Alonso V.Potential impact of the resistance to quaternary ammonium disinfectants on the persistence of Listeria monocytogenes in food processing environments[J]. Frontiers in Microbiology, 2016, 7(3547):1-8.
[6] Xu D, Li Y, Zahid MSH, et al.Benzalkonium chloride and heavy-metal tolerance in Listeria monocytogenes from retail foods[J]. International Journal of Food Microbiology, 2014, 190(3):24-30.
[7] Piddock LJ.Multidrug-resistance efflux pumps - not just for resistance[J]. Nature Reviews Microbiology, 2006, 4(8):629.
[8] Guérin F, Galimand M, Tuambilangana F, et al.Overexpression of the novel MATE fluoroquinolone efflux pump FepA in Listeria monocytogenes is driven by inactivation of its local repressor FepR[J]. PLoS One, 2014, 9(9):e106340.
[9] Huillet E, Velge P, Vallaeys T, et al.LadR, a new PadR-related transcriptional regulator from Listeria monocytogenes, negatively regulates the expression of the multidrug efflux pump MdrL[J]. Fems Microbiology Letters, 2006, 254(1):87-94.
[10] 姜晓冰, 于涛, 牛亚冰, 等. 单核细胞增生李斯特菌喹诺酮耐药机制研究[J]. 生物技术通报, 2016, 32(7):234-241.
[11] Zhao C, Zhao J, Wang W, et al. Expression of MLAA34-HSP70 fusion gene constructed by SOE-PCR[J]. Pakistan Journal of Pharmaceutical Sciences, 2017, 30(3(Special)):1125-1127.
[12] 郭亮, 陈国薇, 谢曼曼, 等. 单增李斯特菌prfA基因缺失菌株的构建及其生物学特性鉴定[J]. 食品科学, 2017, 38(10):12-17.
[13] Bergholz TM, Tang S, Wiedmann M, et al.Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR[J]. Apply Environment Microbiology, 2013, 79(18):5682-5688.
[14] 姜晓冰. 大肠杆菌质粒介导喹诺酮耐药机制研究[D]. 广州:华南理工大学, 2013.
[15] Chen WY, Liu DD, Jian-Hua XU, et al.Application of image J software in analyzing protein expression of recombinant plasmid pET32a-CDK2[J]. China Tropical Medicine, 2014, 1:23-25.
[16] Priyadarshi P, Dravid P, Sheikh I H, et al.Characterization of immunoreactive proteins of Setaria cervi isolated by preparative polyacrylamide gel electrophoresis[J]. Acta Parasitologica, 2017, 62(1):29-37.
[17] Förster-Fromme K, Jendrossek D.AtuR is a repressor of acyclic terpene utilization(Atu)gene cluster expression and specifically binds to two 13 bp inverted repeat sequences of the atuA- atuR intergenic region[J]. Fems Microbiology Letters, 2010, 308(2):166-174
[18] Tamburro M, Ripabelli G, Vitullo M, et al.Gene expression in Listeria monocytogenes exposed to sublethal concentration of benzalkonium chloride[J]. Comparative Immunology Microbiology & Infectious Diseases, 2015, 40:31-39.
[19] Romanova NA, Wolffs PFG, Brovko LY, Griffiths MW.Role of efflux pump in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride[J]. Applied Environmental Microbiology, 2006, 72(5):3498-3503.
[20] Romanova N, Favrin S, Grifiths MW.Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry[J]. Appl Environ Microbiol, 2002, 68:6405-6409.
[21] Hellman LM, Fried MG.Electrophoretic mobility shift assay(EMSA)for detecting protein|[ndash]|nucleic acid interactions[J]. Nature Protocols, 2007, 2(8):1849-1861.