Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity

doi:10.13560/j.cnki.biotech.bull.1985.2018-0861

Abstract

Abstract: To investigate the effect of amino acid mutation in Listeria monocytogenes（LM）ribonuclease RnaseIII RncS on RNA degradation activity,bioinformatics software was used to analyze the domains of RnaseIII encoded by rncS gene of LM wild-type strain SB5,and gene overlap extension PCR（SOE-PCR）was to perform its gene mutations. Then the rncS mutant gene fragments D50A and E122A were cloned into the expression vector pET-32a（+）,and induced by IPTG in Escherichia coli to generate mutant RnaseⅢ. SDS-PAGE and Western Blot were adapted to detect the expression form and the antigen specificity of fusion protein,respectively. Finally,the enzyme activity assay in vitro was to determine the effect of amino acid mutation on RNA degradation activity. The domain analysis revealed that the LM-RnaseIII protein amino acid sequence included a nuclease domain（RIBOc）,in which there were 5 active sites,and a double-stranded RNA binding domain（DSRM）. The results of SDS-PAGE showed that the relative molecular masses of the recombinant mutants RnaseIII-D50A and RnaseIII-E122 A were 42.5 kD,which was consistent with the theoretical values. Western Blot showed that the recombinant mutant RnaseIII-D50A and RnaseIII-E122A reacted with positive serum against LM. Enzyme activity experiments demonstrated that the degradation activity of RnaseIII was dependent on Mn²⁺ or Mg²⁺. After the 50th aspartic acid was mutated,the degradation activity of RnaseIII RncS reduced（P <0.001）,while it significantly（P <0.0001）decreased after the mutation of the 122nd glutamate,suggesting that the 122nd glutamate was the key site for maintaining the activity of LM RnaseIII. This study provides a scientific basis for further revealing the characteristics and mechanism of LM RncS.

Key words: Listeria monocytogenes, ribonuclease Rnase Ⅲ, rncS gene, degradation activity

WANG Li-xia, MENG Qing-ling, QIAO Jun, CAI Kuo-jun, WANG Deng-feng, WU Ye-hui, GUO Jing, CAI Xue-peng. Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity[J]. Biotechnology Bulletin, 2019, 35(3): 110-116.

References

[1] Radoshevich L, Cossart P.Listeria monocytogenes:towards a complete picture of its physiology and pathogenesis.[J]. Nature Reviews Microbiology, 2018, 16(1):32.
[2] Gandhi M, Chikindas ML.Listeria:A foodborne pathogen that knows how to survive.[J]. International Journal of Food Microbiology, 2007, 113(1):1-15.
[3] 孙静娟, 邱景璇, 曾海娟, 等. 单增李斯特菌CdaA的抗原表位分析及抗体的制备[J]. 生物技术通报, 2018, 34(5):163-171.
[4] Neuhaus K, Satorhelyi P, Schauer K, et al.Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans[J]. BMC Genomics, 2013, 27(14):285.
[5] Galié S, Garcíagutiérrez C, Miguélez EM, et al.Biofilms in the food industry:Health aspects and control methods[J]. Frontiers in Microbiology, 2018, 9:898.
[6] Van d VS, Van SS, Molenaar D, et al. The SOS response of Listeria
monocytogenes is involved in stress resistance and mutagenesis.[J]. Microbiology, 2010, 156(2):374-384.
[7] Grubaugh D, Regeimbal JR, Ghosh P, et al. The VirAB ABC transporter is required for VirR regulation of Listeria monocytogenes virulence and resistance to nisin[J]. Infection & Immol/Lunity, 2018, 86(3):IAI. 00901-17.
[8] 张再超, 彭叶龙, 孟庆玲, 等. 单核细胞增生李斯特菌ncRNA研究进展[J]. 生命的化学, 2013, 53(4):87-91.
[9] Arraiano CM, Andrade JM, Domingues S, et al.The critical role of RNA processing and degradation in the control of gene expression[J]. Fems Microbiology Reviews, 2010, 34(5):883-923.
[10] Afonyushkin T, Večerek B, Moll I, et al.Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB[J]. Nucleic Acids Research, 2005, 33(5):1678-1689.
[11] 王立霞, 孟庆玲, 蔡扩军, 等. 单核细胞增生李斯特菌核糖核酸酶RNaseⅢ RncS的表达及其生物学活性研究[J]. 中国预防兽医学报, 2018(7):581-585.
[12] Wagner EG, Altuvia S, Romby P.Antisense RNAs in bacteria and their genetic elements[J]. Advances in Genetics, 2002, 46(3):361-398.
[13] Darfeuille F, Unoson C, Vogel J, et al.An antisense RNA inhibits translation by competing with standby ribosomes[J]. Molecular Cell, 2007, 26(3):381-392.
[14] Lebreton A, Cossart P.RNA-and protein-mediated control of Listeria monocytogenes virulence gene expression[J]. RNA Biol. 2017, 14(5):460-470.
[15] Xiao J, Feehery CE, Tzertzinis G, et al.E. coli RNase III(E38A)generates discrete-sized products from long dsRNA[J]. RNA, 2009, 15(5):984-991.
[16] Blaszczyk J, Tropea JE, Bubunenko M, et al.Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage[J]. Structure, 2001, 9(12):1225-1236.
[17] Sun W, Nicholson AW.Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn²+ rescue of the Glu117Asp mutant[J]. Biochemistry, 2001, 40(16):5102-5110.
[18] Gan J, Tropea JE, Austin BP, et al.Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III[J]. Cell, 2006, 124(2):355-366.
[19] Robertson HD, Webster RE, Zinder ND.A nuclease specific for double-stranded RNA[J]. Virology, 1967, 32(4):718-719.
[20] Blaszczyk J, Gan J, Tropea JE, et al.Noncatalytic assembly of ribonuclease III with double-stranded RNA[J]. Structure, 2004, 12(3):457-466.
[21] Lioliou E, Sharma CM, Caldelari I, et al.Global regulatory functions of the, Staphylococcus aureus, endoribonuclease III in gene expression[J]. PLoS Genetics, 2012, 8(6):3202-3212.
[22] 许先进. 布鲁氏菌核糖核酸酶RibonucleaseⅢ的酶活性研究[D]. 武汉:华中农业大学, 2014.