tuf基因同源超表达对植物乳杆菌LR-1生理行为的影响
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摘要
采用同源超表达及电转化技术,构建植物乳杆菌LR-1的tuf基因超表达重组菌株,对重组菌株的生长情况、抗胁迫能力、黏附能力及生物被膜形成能力进行分析,并检测tuf基因超表达对其他基因的转录水平的影响。结果表明:tuf基因超表达对菌株LR-1的生长情况无非常显著的影响,但可以增强其耐热性能、黏附性能及生物被膜形成能力,且对糖酵解途径参与基因、II型群感关键基因及核糖激酶编码基因fba、pgm、gap、luxS和rib的转录水平具有上调作用。以上结果对乳杆菌的基因功能研究具有参考意义,同时为基因工程菌株在发酵菌剂或益生菌剂的开发应用领域提供了理论支持。
In this study, a recombinant strain expressing the tuf gene of Lactobacillus plantarum LR-1 was constructed by electrotransformation. The growth status, stress resistance, adhesion capacity and biofilm formation of the recombinant strain were evaluated, and the impact of tuf overexpression on the expression of other genes was examined. The results indicated that overexpression of tuf could improve the heat resistance, adhesion capacity and bio?lm formation but did not signi?cantly in?uence the growth of L. plantarum LR-1. Transcriptional analysis suggested that overexpression of tuf could upregulate the expression of genes involved in glycolysis pathway(fba, pgm, and gap), type II quorum sensing system(lux S), and ribose metabolism(rib). Collectively, this study provides useful information for the exploration of gene functions of Lactobacillus and offers a theoretical basis for the application of genetically engineered strains as probiotics or starter cultures.
In this study, a recombinant strain expressing the tuf gene of Lactobacillus plantarum LR-1 was constructed by electrotransformation. The growth status, stress resistance, adhesion capacity and biofilm formation of the recombinant strain were evaluated, and the impact of tuf overexpression on the expression of other genes was examined. The results indicated that overexpression of tuf could improve the heat resistance, adhesion capacity and bio?lm formation but did not signi?cantly in?uence the growth of L. plantarum LR-1. Transcriptional analysis suggested that overexpression of tuf could upregulate the expression of genes involved in glycolysis pathway(fba, pgm, and gap), type II quorum sensing system(lux S), and ribose metabolism(rib). Collectively, this study provides useful information for the exploration of gene functions of Lactobacillus and offers a theoretical basis for the application of genetically engineered strains as probiotics or starter cultures.
引文
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[12]LOPEZ-OCHOA J,MONTES-GARCIA J F,VAZQUEZ C,et al.Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics,participates in cell adhesion,and is present in biofilms[J].Journal of Microbiology,2017,55(9):745-752.DOI:10.1007/s12275-017-7077-0.
[13]NISHIYAMA K,OCHIAI A,TSUBOKAWA D,et al.Identification and characterization of sulfated carbohydrate-binding protein from Lactobacillus reuteri[J].PLoS ONE,2013,8(12):e83703.DOI:10.1371/journal.pone.0083703.
[14]DIANAWATI D,LIM S F,OOI Y,et al.Effect of type of proteinbased microcapsules and storage at various ambient temperatures on the survival and heat tolerance of spray dried Lactobacillus acidophilus[J].Journal of Food Science,2017,82(9):2134-2141.DOI:10.1111/1750-3841.13820.
[15]LAVARI L,BURNS P,PAEZ R,et al.Study of the effects of spray drying in whey-starch on the probiotic capacity of Lactobacillus rhamnosus 64 in the gut of mice[J].Journal of Applied Microbiology,2017,123(4):992-1002.DOI:10.1111/jam.13567.
[16]乔传令.耐热蛋白质延长因子(EF-TU)的分子和功能的性质(英文)[J].实验生物学报,1992(2):165-172.
[17]VERA M,PANI B,GRIFFITHS L A,et al.The translation elongation of eEF1A1 couples transcription to translation during heat shock response[J].Elife,2014,3:e03164.DOI:10.7554/eLife.03164.
[18]MARCOS C M,DE OLIVEIRA H C,DA S J,et al.Identification and characterisation of elongation factor Tu,a novel protein involved in Paracoccidioides brasiliensis-host interaction[J].FEMS Yeast Research,2016,16(7).DOI:10.1093/femsyr/fow079.
[19]YU Y,WANG H,WANG J,et al.Elongation factor thermo unstable(EF-Tu)moonlights as an adhesin on the surface of Mycoplasma hyopneumoniae by binding to fibronectin[J].Frontiers in Microbiology,2018,9:974.DOI:10.3389/fmicb.2018.00974.
[20]CHIU K H,WANG L H,TSAI T T,et al.Secretomic analysis of hostpathogen interactions reveals that elongation factor-Tu is a potential adherence factor of Helicobacter pylori during pathogenesis[J].Journal of Proteome Research,2017,16(1):264-273.DOI:10.1021/acs.jproteome.6b00584.
[21]DHANANI A S,BAGCHI T.The expression of adhesin EF-Tu in response to mucin and its role in Lactobacillus adhesion and competitive inhibition of enteropathogens to mucin[J].Journal of Applied Microbiology,2013,115(2):546-554.DOI:10.1111/jam.12249.
[22]RAMIAH K,VAN REENEN C A,DICKS L M.Expression of the mucus adhesion gene mub,surface layer protein slp and adhesionlike factor EF-TU of Lactobacillus acidophilus ATCC 4356 under digestive stress conditions,as monitored with Real-Time PCR[J].Probiotics and Antimicrobial Proteins,2009,1(1):91.DOI:10.1007/s12602-009-9009-8.
[23]RAMIAH K,VAN REENEN C A,DICKS L M.Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis[J].Research in Microbiology,2008,159(6):470-475.DOI:10.1016/j.resmic.2008.06.002.
[24]刘蕾,武瑞赟,李军,等.基于AI-2介导的类植物乳杆菌生物被膜态与浮游态抗胁迫能力比较与分析[J].食品科学,2017,38(22):41-47.DOI:10.7506/spkx1002-6630-201722007.
[25]刘蕾,武瑞赟,李军,等.类植物乳杆菌L-ZS9生物被膜态的黏附能力及形成影响因素[J].食品科学,2016,37(15):136-143.DOI:10.7506/spkx1002-6630-201615023.
[26]LIU L,WU R,ZHANG J,et al.D-Ribose interferes with quorum sensing to inhibit biofilm formation of Lactobacillus paraplantarum L-ZS9[J].Frontiers in Microbiology,2017,8:1860.DOI:10.3389/fmicb.2017.01860.
[27]LI Z,CHEN Y,LIU D,et al.Involvement of glycolysis/gluconeogenesis and signaling regulatory pathways in Saccharomyces cerevisiae biofilms during fermentation[J].Frontiers in Microbiology,2015,6:139.DOI:10.3389/fmicb.2015.00139.
[28]ZHOU Q,FENG X,ZHANG Q,et al.Carbon catabolite control is important for Listeria monocytogenes biofilm formation in response to nutrient availability[J].Current Opinion in Microbiology,2012,65(1):35-43.DOI:10.1007/s00284-012-0125-4.
[29]YARAWSKY A E,ENGLISH L R,WHITTEN S T,et al.The proline/glycine-rich region of the biofilm adhesion protein aap forms an extended stalk that resists compaction[J].Journal of Molecular Biology,2017,429(2):261-279.DOI:10.1016/j.jmb.2016.11.017.
[30]BERLUTTI F,MOREA C,BATTISTONI A,et al.Iron availability influences aggregation,biofilm,adhesion and invasion of Pseudomonas aeruginosa and Burkholderia cenocepacia[J].International Journal of Immunopathology and Pharmacology,2005,18(4):661-670.DOI:10.1177/039463200501800407.
[31]JIA F F,ZHENG H Q,SUN S R,et al.Role of luxS in stress tolerance and adhesion ability in Lactobacillus plantarum KLDS1.0391[J].Biomed Research International,2018,2018:4506829.DOI:10.1155/2018/4506829.
[32]JU X,LI J,ZHU M,et al.Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin[J].Food Research International,2018,107:385-393.DOI:10.1016/j.foodres.2018.02.039.
[33]JIA F F,PANG X H,ZHU D Q,et al.Role of the luxS gene in bacteriocin biosynthesis by Lactobacillus plantarum KLDS1.0391:a proteomic analysis[J].Scientific Reports,2017,7(1):13871.DOI:10.1038/s41598-017-13231-4.
[34]BUCK B L,AZCARATE-PERIL M A,KLAENHAMMER T R.Role of autoinducer-2 on the adhesion ability of Lactobacillus acidophilus[J].Journal of Applied Microbiology,2009,107(1):269-279.DOI:10.1111/j.1365-2672.2009.04204.x.
[35]LEBEER S,CLAES I J,VERHOEVEN T L,et al.Impact of luxS and suppressor mutations on the gastrointestinal transit of Lactobacillus rhamnosus GG[J].Applied and Environmental Microbiology,2008,74(15):4711-4718.DOI:10.1128/AEM.00133-08.
[36]TANNOCK G W,GHAZALLY S,WALTER J,et al.Ecological behavior of Lactobacillus reuteri 100-23 is affected by mutation of luxS gene[J].Applied and Environmental Microbiology,2005,71(12):8419-8425.DOI:10.1128/AEM.71.12.8419-8425.2005.
[37]MOSLEHI-JENABIAN S,GORI K,JESPERSEN L.AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp.[J].International Journal of Food Microbiology,2009,135(3):295-302.DOI:10.1016/j.ijfoodmicro.2009.08.011.
[2]ATHIYYAH A F,DARMA A,RANUH R,et al.Lactobacillus plantarum IS-10506 activates intestinal stem cells in a rodent model[J].Beneficial Microbes,2018,9(5):755-760.DOI:10.3920/BM2017.0118.
[3]KIM K,LEE G,THANH H D,et al.Exopolysaccharide from Lactobacillus plantarum LRCC5310 offers protection against rotavirus-induced diarrhea and regulates inflammatory response[J].Journal of Dairy Science,2018,101(7):5702-5712.DOI:10.3168/jds.2017-14151.
[4]MOKHTARI S,JAFARI S M,KHOMEIRI M,et al.The cell wall compound of Saccharomyces cerevisiae as a novel wall material for encapsulation of probiotics[J].Food Research International,2017,96:19-26.DOI:10.1016/j.foodres.2017.03.014.
[5]JIANG Y,ZHENG Z,ZHANG T,et al.Microencapsulation of Lactobacillus acidophilus NCFM using polymerized whey proteins as wall material[J].International Journal of Food Sciences and Nutrition,2016,67(6):670-677.DOI:10.1080/09637486.2016.1194810.
[6]WIDJAJA M,HARVEY K L,HAGEMANN L,et al.Elongation factor Tu is a multifunctional and processed moonlighting protein[J].Scientific Reports,2017,7(1):11227.DOI:10.1038/s41598-017-10644-z.
[7]PENG Z,VOGEL R F,EHRMANN M A,et al.Identification and characterization of adhesion proteins in Lactobacilli targeting actin as receptor[J].Molecular and Cellular Probes,2018,37:60-63.DOI:10.1016/j.mcp.2017.08.002.
[8]SHARMA J,MISHRA B B,LI Q,et al.TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu[J].Cellular Immunology,2011,269(2):69-73.DOI:10.1016/j.cellimm.2011.03.023.
[9]WOLFF D G,CASTIBLANCO-VALENCIA M M,ABE C M,et al.Interaction of Leptospira elongation factor Tu with plasminogen and complement factor H:a metabolic leptospiral protein with moonlighting activities[J].PLoS ONE,2013,8(11):e81818.DOI:10.1371/journal.pone.0081818.
[10]HE Y,WANG H,CHEN L.Comparative secretomics reveals novel virulence-associated factors of Vibrio parahaemolyticus[J].Frontiers in Microbiology,2015,6:707.DOI:10.3389/fmicb.2015.00707.
[11]PAPA R,ARTINI M,CELLINI A,et al.A new anti-infective strategy to reduce the spreading of antibiotic resistance by the action on adhesionmediated virulence factors in Staphylococcus aureus[J].Microbial Pathogenesis,2013,63:44-53.DOI:10.1016/j.micpath.2013.05.003.
[12]LOPEZ-OCHOA J,MONTES-GARCIA J F,VAZQUEZ C,et al.Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics,participates in cell adhesion,and is present in biofilms[J].Journal of Microbiology,2017,55(9):745-752.DOI:10.1007/s12275-017-7077-0.
[13]NISHIYAMA K,OCHIAI A,TSUBOKAWA D,et al.Identification and characterization of sulfated carbohydrate-binding protein from Lactobacillus reuteri[J].PLoS ONE,2013,8(12):e83703.DOI:10.1371/journal.pone.0083703.
[14]DIANAWATI D,LIM S F,OOI Y,et al.Effect of type of proteinbased microcapsules and storage at various ambient temperatures on the survival and heat tolerance of spray dried Lactobacillus acidophilus[J].Journal of Food Science,2017,82(9):2134-2141.DOI:10.1111/1750-3841.13820.
[15]LAVARI L,BURNS P,PAEZ R,et al.Study of the effects of spray drying in whey-starch on the probiotic capacity of Lactobacillus rhamnosus 64 in the gut of mice[J].Journal of Applied Microbiology,2017,123(4):992-1002.DOI:10.1111/jam.13567.
[16]乔传令.耐热蛋白质延长因子(EF-TU)的分子和功能的性质(英文)[J].实验生物学报,1992(2):165-172.
[17]VERA M,PANI B,GRIFFITHS L A,et al.The translation elongation of eEF1A1 couples transcription to translation during heat shock response[J].Elife,2014,3:e03164.DOI:10.7554/eLife.03164.
[18]MARCOS C M,DE OLIVEIRA H C,DA S J,et al.Identification and characterisation of elongation factor Tu,a novel protein involved in Paracoccidioides brasiliensis-host interaction[J].FEMS Yeast Research,2016,16(7).DOI:10.1093/femsyr/fow079.
[19]YU Y,WANG H,WANG J,et al.Elongation factor thermo unstable(EF-Tu)moonlights as an adhesin on the surface of Mycoplasma hyopneumoniae by binding to fibronectin[J].Frontiers in Microbiology,2018,9:974.DOI:10.3389/fmicb.2018.00974.
[20]CHIU K H,WANG L H,TSAI T T,et al.Secretomic analysis of hostpathogen interactions reveals that elongation factor-Tu is a potential adherence factor of Helicobacter pylori during pathogenesis[J].Journal of Proteome Research,2017,16(1):264-273.DOI:10.1021/acs.jproteome.6b00584.
[21]DHANANI A S,BAGCHI T.The expression of adhesin EF-Tu in response to mucin and its role in Lactobacillus adhesion and competitive inhibition of enteropathogens to mucin[J].Journal of Applied Microbiology,2013,115(2):546-554.DOI:10.1111/jam.12249.
[22]RAMIAH K,VAN REENEN C A,DICKS L M.Expression of the mucus adhesion gene mub,surface layer protein slp and adhesionlike factor EF-TU of Lactobacillus acidophilus ATCC 4356 under digestive stress conditions,as monitored with Real-Time PCR[J].Probiotics and Antimicrobial Proteins,2009,1(1):91.DOI:10.1007/s12602-009-9009-8.
[23]RAMIAH K,VAN REENEN C A,DICKS L M.Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis[J].Research in Microbiology,2008,159(6):470-475.DOI:10.1016/j.resmic.2008.06.002.
[24]刘蕾,武瑞赟,李军,等.基于AI-2介导的类植物乳杆菌生物被膜态与浮游态抗胁迫能力比较与分析[J].食品科学,2017,38(22):41-47.DOI:10.7506/spkx1002-6630-201722007.
[25]刘蕾,武瑞赟,李军,等.类植物乳杆菌L-ZS9生物被膜态的黏附能力及形成影响因素[J].食品科学,2016,37(15):136-143.DOI:10.7506/spkx1002-6630-201615023.
[26]LIU L,WU R,ZHANG J,et al.D-Ribose interferes with quorum sensing to inhibit biofilm formation of Lactobacillus paraplantarum L-ZS9[J].Frontiers in Microbiology,2017,8:1860.DOI:10.3389/fmicb.2017.01860.
[27]LI Z,CHEN Y,LIU D,et al.Involvement of glycolysis/gluconeogenesis and signaling regulatory pathways in Saccharomyces cerevisiae biofilms during fermentation[J].Frontiers in Microbiology,2015,6:139.DOI:10.3389/fmicb.2015.00139.
[28]ZHOU Q,FENG X,ZHANG Q,et al.Carbon catabolite control is important for Listeria monocytogenes biofilm formation in response to nutrient availability[J].Current Opinion in Microbiology,2012,65(1):35-43.DOI:10.1007/s00284-012-0125-4.
[29]YARAWSKY A E,ENGLISH L R,WHITTEN S T,et al.The proline/glycine-rich region of the biofilm adhesion protein aap forms an extended stalk that resists compaction[J].Journal of Molecular Biology,2017,429(2):261-279.DOI:10.1016/j.jmb.2016.11.017.
[30]BERLUTTI F,MOREA C,BATTISTONI A,et al.Iron availability influences aggregation,biofilm,adhesion and invasion of Pseudomonas aeruginosa and Burkholderia cenocepacia[J].International Journal of Immunopathology and Pharmacology,2005,18(4):661-670.DOI:10.1177/039463200501800407.
[31]JIA F F,ZHENG H Q,SUN S R,et al.Role of luxS in stress tolerance and adhesion ability in Lactobacillus plantarum KLDS1.0391[J].Biomed Research International,2018,2018:4506829.DOI:10.1155/2018/4506829.
[32]JU X,LI J,ZHU M,et al.Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin[J].Food Research International,2018,107:385-393.DOI:10.1016/j.foodres.2018.02.039.
[33]JIA F F,PANG X H,ZHU D Q,et al.Role of the luxS gene in bacteriocin biosynthesis by Lactobacillus plantarum KLDS1.0391:a proteomic analysis[J].Scientific Reports,2017,7(1):13871.DOI:10.1038/s41598-017-13231-4.
[34]BUCK B L,AZCARATE-PERIL M A,KLAENHAMMER T R.Role of autoinducer-2 on the adhesion ability of Lactobacillus acidophilus[J].Journal of Applied Microbiology,2009,107(1):269-279.DOI:10.1111/j.1365-2672.2009.04204.x.
[35]LEBEER S,CLAES I J,VERHOEVEN T L,et al.Impact of luxS and suppressor mutations on the gastrointestinal transit of Lactobacillus rhamnosus GG[J].Applied and Environmental Microbiology,2008,74(15):4711-4718.DOI:10.1128/AEM.00133-08.
[36]TANNOCK G W,GHAZALLY S,WALTER J,et al.Ecological behavior of Lactobacillus reuteri 100-23 is affected by mutation of luxS gene[J].Applied and Environmental Microbiology,2005,71(12):8419-8425.DOI:10.1128/AEM.71.12.8419-8425.2005.
[37]MOSLEHI-JENABIAN S,GORI K,JESPERSEN L.AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp.[J].International Journal of Food Microbiology,2009,135(3):295-302.DOI:10.1016/j.ijfoodmicro.2009.08.011.