溶藻弧菌ZJ-T小RNA srvg23535基因突变株的构建及其功能初探
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摘要
【背景】小RNA被证实对细菌遗传变异、生长繁殖、致病性等具有重要的调控作用,但是在机会致病菌——溶藻弧菌(Vibrioalginolyticus)中研究较少。本实验室前期通过转录组测序和Northern Blot鉴定得到溶藻弧菌ZJ-T中小RNA srvg23535。【目的】初探小RNA srvg23535对溶藻弧菌生物学特性的影响。【方法】利用同源重组技术构建小RNA srvg23535的缺失突变株,并对野生株和srvg23535突变株的菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及生长代谢等生物学特性进行比较研究。【结果】通过对溶藻弧菌小RNA srvg23535基因缺失突变株的生物学特性分析表明,小RNA srvg23535缺失后,对溶藻弧菌菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及多数测定的碳源、氮源代谢的影响不显著;但突变株对D-海藻糖(D-trehalose)的代谢减弱,对果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)的代谢增强,并可利用丙氨酸-天冬氨酸(Ala-Asp)为氮源。【结论】小RNA srvg23535参与调控溶藻弧菌对D-海藻糖(D-trehalose)、果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)和丙氨酸-天冬氨酸(Ala-Asp)的代谢,这将为获取srvg23535调控靶标基因,进而阐明其与靶标基因的相互作用机制奠定基础。
[Background] Small non-coding RNAs(sRNAs) play important roles in genetic variation,growth, reproduction, and bacterial pathogenicity, but few studies have been conducted on the opportunistic pathogen of Vibrio alginolyticus. We have identified an sRNA, srvg23535, of V.alginolyticus ZJ-T by RNA-seq and Northern Blot. [Objective] We studied the effect of srvg23535 on the biological characteristics of V. alginolyticus. [Methods] The srvg23535 knock-out mutant was constructed by homologous recombination. The function of srvg23535 was studied with comparing the colony morphology, motility, extracellular protease secretion, pressure sensing of H_2O_2 and Cu~(2+), iron utilization,antibiotic resistance, and metabolism between the wild type and the mutant strains. [Results] The biological characteristics analysis of srvg23535 knock-out mutant showed that the absence of srvg23535 had no significant effect on the colony morphology, motility, extracellular protease secretion, pressure sensing of H_2O_2 and Cu~(2+), iron utilization, antibiotic resistance and the metabolism of most of the tested carbon and nitrogen sources in V. alginolyticus. However, the mutant showed a weaker metabolism of D-trehalose, a stronger metabolism of pectin and alanine-glutamine(Ala-Gln), and accessibility to the utilization of alanine-aspartic acid(Ala-Asp) as the sole nitrogen source. [Conclusion] The sRNA srvg23535 is involved in the metabolism regulation of D-trehalose, pectin, Ala-Gln and Ala-Asp with laying the foundation for the target genes screening of srvg23535 and further elucidating the regulation mechanism of srvg23535 to its target genes.
[Background] Small non-coding RNAs(sRNAs) play important roles in genetic variation,growth, reproduction, and bacterial pathogenicity, but few studies have been conducted on the opportunistic pathogen of Vibrio alginolyticus. We have identified an sRNA, srvg23535, of V.alginolyticus ZJ-T by RNA-seq and Northern Blot. [Objective] We studied the effect of srvg23535 on the biological characteristics of V. alginolyticus. [Methods] The srvg23535 knock-out mutant was constructed by homologous recombination. The function of srvg23535 was studied with comparing the colony morphology, motility, extracellular protease secretion, pressure sensing of H_2O_2 and Cu~(2+), iron utilization,antibiotic resistance, and metabolism between the wild type and the mutant strains. [Results] The biological characteristics analysis of srvg23535 knock-out mutant showed that the absence of srvg23535 had no significant effect on the colony morphology, motility, extracellular protease secretion, pressure sensing of H_2O_2 and Cu~(2+), iron utilization, antibiotic resistance and the metabolism of most of the tested carbon and nitrogen sources in V. alginolyticus. However, the mutant showed a weaker metabolism of D-trehalose, a stronger metabolism of pectin and alanine-glutamine(Ala-Gln), and accessibility to the utilization of alanine-aspartic acid(Ala-Asp) as the sole nitrogen source. [Conclusion] The sRNA srvg23535 is involved in the metabolism regulation of D-trehalose, pectin, Ala-Gln and Ala-Asp with laying the foundation for the target genes screening of srvg23535 and further elucidating the regulation mechanism of srvg23535 to its target genes.
引文
[1]Siddiqui R,Alam MM,Naser MN,et al.Prevalence of Vibrio alginolyticus in sediment samples of river and coastal areas of Bangladesh[J].Bangladesh Journal of Microbiology,2016,29(1):1-6
[2]Citil BE,Derin S,Sankur F,et al.Vibrio alginolyticus associated chronic myringitis acquired in Mediterranean waters of Turkey[J].Case Reports in Infectious Diseases,2015,2015(42):187212-187214
[3]Zheng TL,Wang GL,Jin S,et al.Studies on pathogen of ulcer disease of cultured large yellow croakers,Pseudosciaena crocea and its control medicines[J].South China Fisheries Science,2006,2(1):5-10(in Chinese)郑天伦,王国良,金珊,等.养殖大黄鱼溃疡病的病原菌及其防治药物研究[J].南方水产科学,2006,2(1):5-10
[4]Newton A,Mahon BE.Increasing rates of Vibriosis in the United States,1996-2010:review of surveillance data from 2systems[J].Clinical Infectious Diseases,2012,54(s5):S391-395
[5]Baran I,Acar A,Gen?Y,et al.Case report:A case of otitis externa due to Vibrio alginolyticus[J].Türk Hijiyen Ve Deneysel Biyoloji Dergisi Turkish Bulletin of Hygiene&Experimental Biology,2016,73(1):49-54
[6]Gaüzère BA,Chanareille P,Vandroux D.Post nearly drowning Vibrio alginolyticus septicemia acquired in reunion(Indian ocean)][J].Bulletin De La Societe De Pathologie Exotique,2016,109(3):151-154
[7]Chen C,Xie J,Hu C.Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus[J].Biofouling,2009,25(6):525-531
[8]Solano C,Echeverz M,Lasa I.Biofilm dispersion and quorum sensing[J].Current Opinion in Microbiology,2014,18(4):96-104
[9]Alonso CR.Post-transcriptional gene regulation via RNAcontrol[M].Humana Press,2008
[10]Wang Z,Sun X,Zhao Y,et al.Evolution of gene regulation during transcription and translation[J].Genome Biology&Evolution,2015,7(4):1155-1167
[11]Storz G,Vogel J,Wassarman KM.Regulation by small RNAs in bacteria:expanding frontiers[J].Molecular Cell,2011,43(6):880-891
[12]Lenz DH,Miller M B,Zhu J,et al.CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae[J].Molecular Microbiology,2005,58(4):1186-1202
[13]Liu JM,Camilli A.A broadening world of bacterial small RNAs[J].Current Opinion in Microbiology,2010,13(1):18-23
[14]Toledo-Arana A,Repoila F,Cossart P.Small noncoding RNAs controlling pathogenesis[J].Current Opinion in Microbiology,2007,10(2):182-188
[15]Bohn C,Rigoulay C,Chabelskaya S,et al.Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism[J].Nucleic Acids Research,2010,38(19):6620-6636
[16]Sharma CM,Hoffmann S,Darfeuille F,et al.The primary transcriptome of the major human pathogen Helicobacter pylori[J].Nature,2010,464(7286):250-255
[17]Huang L,Hu J,Su Y,et al.Identification and characterization of three Vibrio alginolyticus non-coding RNAs involved in adhesion,chemotaxis,and motility processes[J].Frontiers in Cellular&Infection Microbiology,2015,5:56-68
[18]Huang L,Hu J,Su Y,et al.Genome-wide detection of predicted non-coding RNAs related to the adhesion process in Vibrio alginolyticus using high-throughput sequencing[J].Frontiers in Microbiology,2016,7:619-628
[19]Huang X,Chen C,Ren C,et al.Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51[J].Biofouling,2017,34(1):1-14
[20]Val ME,Skovgaard O,Ducos-galand M,et al.Genome engineering in Vibrio cholerae:a feasible approach to address biological issues[J].PLoS Genetics,2012,8(1):e1002472
[21]Le RF,Binesse J,Saulnier D,et al.Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector[J].Applied and Environmental Microbiology,2007,73(3):777-784
[22]Nguyen AN,Disconzi E,Charrière GM,et al.csrB gene duplication drives the evolution of redundant regulatory pathways controlling expression of the major toxic secreted metalloproteases in Vibrio tasmaniensis LGP32[J].m Sphere,2018,3(6):e00582-18
[23]Deng YQ,Chen C,Zhao Z,et al.The RNA chaperone Hfq is involved in colony morphology,nutrient utilization and oxidative and envelope stress response in Vibrio alginolyticus[J].PLoSOne,2016,11(9):e0163689
[24]Deng YQ,Zhao Z,Liu SL,et al.Preliminary research of effect of different carbon sources on adhesion-related phenotypes in Vibrio alginolyticus[J].Marine Sciences,2016,40(11):99-107(in Chinese)邓益琴,赵哲,刘松林,等.不同碳源对溶藻弧菌黏附相关表型的影响初探及Hfq对其的调控[J].海洋科学,2016,40(11):99-107
[25]Deng YQ,Zhao JJ,Liu SL,et al.Role of hfq in virulence regulation of Vibrio alginolyticus[J].Oceanologia Et Limnologia Sinica,2016,47(3):604-611(in Chinese)邓益琴,赵晶晶,刘松林,等.Hfq对溶藻弧菌(Vibrio alginolyticus)毒力的调控分析[J].海洋与湖沼,2016,47(3):604-611
[26]Bochner BR,Gadzinski P,Panomitros E.Phenotype microarrays for high-throughput phenotypic testing and assay of gene function[J].Genome Research,2001,11(7):1246-1255
[27]Ahmad S,Ahmad M,Khan S,et al.An overview on phase variation,mechanisms and roles in bacterial adaptation[J].Jpma the Journal of the Pakistan Medical Association,2017,67(2):285-291
[28]Vogel J.A rough guide to the non‐coding RNA world of Salmonella[J].Molecular Microbiology,2009,71(1):1-11
[29]Wagner EGH,Romby P.Small RNAs in bacteria and archaea:who they are,what they do,and how they do it[J].Advances in Genetics,2015,90:133-208
[30]Urbanowski ML,Stauffer LT,Stauffer GV.The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli[J].Molecular Microbiology,2000,37(4):856-868
[31]Heidrich N,Moll I,Brantl S.In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrCm RNA[J].Nucleic Acids Research,2007,35(13):4331-4346
[32]Gimpel M,Heidrich N,M?der U,et al.A dual-function sRNAfrom Bacillus subtilis:SR1 acts as a peptide encoding mRNA on the gapA operon[J].Molecular Microbiology,2010,76(4):990-1009
[33]Nguyen AN,Jacq A.Small RNAs in the Vibrionaceae:an ocean still to be explored[J].Wiley Interdisciplinary Reviews Rna,2014,5(3):381-392
[34]Kai P,Pfeiffer V,Mika F,et al.σE‐dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp m RNA decay[J].Molecular Microbiology,2006,62(6):1674-1688
[35]MasséE,Gottesman S.A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(7):4620-4625
[36]Revelles O,Millard P,Nougayrède JP,et al.The carbon storage regulator(Csr)system exerts a nutrient-specific control over central metabolism in Escherichia coli strain Nissle 1917[J].PLoS One,2013,8(6):e66386
[37]Becker A,Schl?der P,Steele JE,et al.The regulation of trehalose metabolism in insects[J].Experientia,1996,52(5):433-439
[38]Attfield PV.Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response[J].FEBS Letters,1987,225(1-2):259-263
[39]Benaroudj N,Lee DH,Goldberg AL.Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals[J].Journal of Biological Chemistry,2001,276(26):24261-24267
[40]Kandror O,Deleon A,Goldberg AL.Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(15):9727-9732
[41]Deng Y,Chen C,Zhao Z,et al.Complete Genome Sequence of Vibrio alginolyticus ZJ-T[J].Genome Announcements,2016,4(5):e00912-16
[42]Pérez S,Mazeau K,Penhoat CHD.The three-dimensional structures of the pectic polysaccharides[J].Plant Physiology&Biochemistry,2000,38(1-2):37-55
[43]Mohnen D.Pectin structure and biosynthesis[J].Current Opinion in Plant Biology,2008,11(3):266-277
[44]Urban JH,Vogel J.Translational control and target recognition by Escherichia coli small RNAs in vivo[J].Nucleic Acids Research,2007,35(3):1018-1037
[2]Citil BE,Derin S,Sankur F,et al.Vibrio alginolyticus associated chronic myringitis acquired in Mediterranean waters of Turkey[J].Case Reports in Infectious Diseases,2015,2015(42):187212-187214
[3]Zheng TL,Wang GL,Jin S,et al.Studies on pathogen of ulcer disease of cultured large yellow croakers,Pseudosciaena crocea and its control medicines[J].South China Fisheries Science,2006,2(1):5-10(in Chinese)郑天伦,王国良,金珊,等.养殖大黄鱼溃疡病的病原菌及其防治药物研究[J].南方水产科学,2006,2(1):5-10
[4]Newton A,Mahon BE.Increasing rates of Vibriosis in the United States,1996-2010:review of surveillance data from 2systems[J].Clinical Infectious Diseases,2012,54(s5):S391-395
[5]Baran I,Acar A,Gen?Y,et al.Case report:A case of otitis externa due to Vibrio alginolyticus[J].Türk Hijiyen Ve Deneysel Biyoloji Dergisi Turkish Bulletin of Hygiene&Experimental Biology,2016,73(1):49-54
[6]Gaüzère BA,Chanareille P,Vandroux D.Post nearly drowning Vibrio alginolyticus septicemia acquired in reunion(Indian ocean)][J].Bulletin De La Societe De Pathologie Exotique,2016,109(3):151-154
[7]Chen C,Xie J,Hu C.Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus[J].Biofouling,2009,25(6):525-531
[8]Solano C,Echeverz M,Lasa I.Biofilm dispersion and quorum sensing[J].Current Opinion in Microbiology,2014,18(4):96-104
[9]Alonso CR.Post-transcriptional gene regulation via RNAcontrol[M].Humana Press,2008
[10]Wang Z,Sun X,Zhao Y,et al.Evolution of gene regulation during transcription and translation[J].Genome Biology&Evolution,2015,7(4):1155-1167
[11]Storz G,Vogel J,Wassarman KM.Regulation by small RNAs in bacteria:expanding frontiers[J].Molecular Cell,2011,43(6):880-891
[12]Lenz DH,Miller M B,Zhu J,et al.CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae[J].Molecular Microbiology,2005,58(4):1186-1202
[13]Liu JM,Camilli A.A broadening world of bacterial small RNAs[J].Current Opinion in Microbiology,2010,13(1):18-23
[14]Toledo-Arana A,Repoila F,Cossart P.Small noncoding RNAs controlling pathogenesis[J].Current Opinion in Microbiology,2007,10(2):182-188
[15]Bohn C,Rigoulay C,Chabelskaya S,et al.Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism[J].Nucleic Acids Research,2010,38(19):6620-6636
[16]Sharma CM,Hoffmann S,Darfeuille F,et al.The primary transcriptome of the major human pathogen Helicobacter pylori[J].Nature,2010,464(7286):250-255
[17]Huang L,Hu J,Su Y,et al.Identification and characterization of three Vibrio alginolyticus non-coding RNAs involved in adhesion,chemotaxis,and motility processes[J].Frontiers in Cellular&Infection Microbiology,2015,5:56-68
[18]Huang L,Hu J,Su Y,et al.Genome-wide detection of predicted non-coding RNAs related to the adhesion process in Vibrio alginolyticus using high-throughput sequencing[J].Frontiers in Microbiology,2016,7:619-628
[19]Huang X,Chen C,Ren C,et al.Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51[J].Biofouling,2017,34(1):1-14
[20]Val ME,Skovgaard O,Ducos-galand M,et al.Genome engineering in Vibrio cholerae:a feasible approach to address biological issues[J].PLoS Genetics,2012,8(1):e1002472
[21]Le RF,Binesse J,Saulnier D,et al.Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector[J].Applied and Environmental Microbiology,2007,73(3):777-784
[22]Nguyen AN,Disconzi E,Charrière GM,et al.csrB gene duplication drives the evolution of redundant regulatory pathways controlling expression of the major toxic secreted metalloproteases in Vibrio tasmaniensis LGP32[J].m Sphere,2018,3(6):e00582-18
[23]Deng YQ,Chen C,Zhao Z,et al.The RNA chaperone Hfq is involved in colony morphology,nutrient utilization and oxidative and envelope stress response in Vibrio alginolyticus[J].PLoSOne,2016,11(9):e0163689
[24]Deng YQ,Zhao Z,Liu SL,et al.Preliminary research of effect of different carbon sources on adhesion-related phenotypes in Vibrio alginolyticus[J].Marine Sciences,2016,40(11):99-107(in Chinese)邓益琴,赵哲,刘松林,等.不同碳源对溶藻弧菌黏附相关表型的影响初探及Hfq对其的调控[J].海洋科学,2016,40(11):99-107
[25]Deng YQ,Zhao JJ,Liu SL,et al.Role of hfq in virulence regulation of Vibrio alginolyticus[J].Oceanologia Et Limnologia Sinica,2016,47(3):604-611(in Chinese)邓益琴,赵晶晶,刘松林,等.Hfq对溶藻弧菌(Vibrio alginolyticus)毒力的调控分析[J].海洋与湖沼,2016,47(3):604-611
[26]Bochner BR,Gadzinski P,Panomitros E.Phenotype microarrays for high-throughput phenotypic testing and assay of gene function[J].Genome Research,2001,11(7):1246-1255
[27]Ahmad S,Ahmad M,Khan S,et al.An overview on phase variation,mechanisms and roles in bacterial adaptation[J].Jpma the Journal of the Pakistan Medical Association,2017,67(2):285-291
[28]Vogel J.A rough guide to the non‐coding RNA world of Salmonella[J].Molecular Microbiology,2009,71(1):1-11
[29]Wagner EGH,Romby P.Small RNAs in bacteria and archaea:who they are,what they do,and how they do it[J].Advances in Genetics,2015,90:133-208
[30]Urbanowski ML,Stauffer LT,Stauffer GV.The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli[J].Molecular Microbiology,2000,37(4):856-868
[31]Heidrich N,Moll I,Brantl S.In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrCm RNA[J].Nucleic Acids Research,2007,35(13):4331-4346
[32]Gimpel M,Heidrich N,M?der U,et al.A dual-function sRNAfrom Bacillus subtilis:SR1 acts as a peptide encoding mRNA on the gapA operon[J].Molecular Microbiology,2010,76(4):990-1009
[33]Nguyen AN,Jacq A.Small RNAs in the Vibrionaceae:an ocean still to be explored[J].Wiley Interdisciplinary Reviews Rna,2014,5(3):381-392
[34]Kai P,Pfeiffer V,Mika F,et al.σE‐dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp m RNA decay[J].Molecular Microbiology,2006,62(6):1674-1688
[35]MasséE,Gottesman S.A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(7):4620-4625
[36]Revelles O,Millard P,Nougayrède JP,et al.The carbon storage regulator(Csr)system exerts a nutrient-specific control over central metabolism in Escherichia coli strain Nissle 1917[J].PLoS One,2013,8(6):e66386
[37]Becker A,Schl?der P,Steele JE,et al.The regulation of trehalose metabolism in insects[J].Experientia,1996,52(5):433-439
[38]Attfield PV.Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response[J].FEBS Letters,1987,225(1-2):259-263
[39]Benaroudj N,Lee DH,Goldberg AL.Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals[J].Journal of Biological Chemistry,2001,276(26):24261-24267
[40]Kandror O,Deleon A,Goldberg AL.Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(15):9727-9732
[41]Deng Y,Chen C,Zhao Z,et al.Complete Genome Sequence of Vibrio alginolyticus ZJ-T[J].Genome Announcements,2016,4(5):e00912-16
[42]Pérez S,Mazeau K,Penhoat CHD.The three-dimensional structures of the pectic polysaccharides[J].Plant Physiology&Biochemistry,2000,38(1-2):37-55
[43]Mohnen D.Pectin structure and biosynthesis[J].Current Opinion in Plant Biology,2008,11(3):266-277
[44]Urban JH,Vogel J.Translational control and target recognition by Escherichia coli small RNAs in vivo[J].Nucleic Acids Research,2007,35(3):1018-1037