摘要
目的
鉴定假肠膜明串珠菌L64基因组中编码的mazEF类II型毒素-抗毒素系统，并初步探讨mazEF系统协助宿主应对环境酸压力的分子机制。
方法
在大肠杆菌中诱导表达MazF毒素，或同时表达其对应的抗毒素蛋白，以检测MazF是否抑制宿主的正常生长，并验证对应的抗毒素是否能中和MazF的毒性。基于lacZ报告系统及电泳迁移率变动分析(electrophoretic mobility shift assay, EMSA)等体内外试验确定mazEF系统的自调控机制。通过生物信息学预测及体内外试验确定受MazE调控的下游基因。
结果
在假肠膜明串珠菌L64基因组编码的3对潜在mazEF毒素-抗毒素系统中，仅有mazEF1-Leup (OYT_01690-OYT_01685)编码真正的毒素-抗毒素系统。抗毒素MazE1-Leup (OYT_01685)通过与启动子中的回文序列(TAACAaaatgTGTTA)结合，抑制mazEF1-Leup启动子的转录；同时，MazE1-Leup通过与dlt-acpS-alr操纵子启动子中的相同回文序列(TAACAtattgaaatatatgTGTTA)结合，抑制dlt-acpS-alr的转录。
结论
OYT_01690-OYT_01685编码一个真正的mazEF类毒素-抗毒素系统，该系统不仅参与自身转录的调控，还抑制dlt-acpS-alr操纵子的转录，从而协助假肠膜明串珠菌L64应对环境中的酸压力。
毒素-抗毒素系统(toxin-antitoxin system, TA system)最初发现于低拷贝质粒中，其通过分裂后自杀机制确保质粒的稳定遗
在II型TA系统中，抗毒素基因通常位于毒素基因的上游，但也有少数II型TA系统呈现出相反的遗传结构，即毒素基因位于抗毒素基因的上游。Li
尽管II型TA系统在其他细菌中，尤其是条件致病菌中得到了广泛研究，但关于乳酸菌，特别是明串珠菌来源的TA系统研究仍相对匮
1 材料与方法
1.1 材料
1.1.1 菌株、质粒及培养条件
本研究所用菌株和质粒的详细信息见
菌株/质粒 Strains/Plasmids | 主要特征 Features | 来源 Origin |
|---|---|---|
| Leuconostoc pseudomesenteroides L64 | Wild type | Lab |
| Escherichia coli TOP10 |
| ThermoFisher Scientific |
| Escherichia coli BL21(DE3) |
| Novagene |
| Escherichia coli BTH101 |
| Euromedex |
| Escherichia coli XL1-Blue |
| ThermoFisher Scientific |
| His-LacZ | pUC ori, araC, bla, promoter-less lacZ | Lab |
| pACYCDuet-1 | P15A ori, lacI, cat, two MCSs | Novegene |
| pBAD/myc-hisA | pBR322 ori, bla, araC, pBAD | Life Tech. |
| pUT18C | pUC ori, T18, bla | Euromedex |
| pKT25 | pUC ori, T25, kan | Euromedex |
| pBAD-F1 | Expression of MazF1-Leup controlled by arabinose inducible promoter | This study |
| pBAD-F2 | Expression of MazF2-Leup controlled by arabinose inducible promoter | This study |
| pBAD-F3 | Expression of MazF3-Leup controlled by arabinose inducible promoter | This study |
| pUT18C-zipper | pUC ori, zipper-T18, bla | Euromedex |
| pKT25-zipper | pUC ori, zipper-T25, kan | Euromedex |
| pPef1-lacZ | Expression of report gene lacZ controlled by promoter of mazEF1-Leup | This study |
| pPef1-lacZ-Pcat-E1 | Simultaneous expression of MazE1-Leup on the basis of pPef1-lacZ | This study |
| pPef1-lacZ-Pcat-EF1 | Simultaneous expression of MazEF1-Leup on the basis of pPef1-lacZ | This study |
| pPef1-IRM-lacZ | Mutation of IR in promoter of mazEF1-Leup | This study |
| pPef1-IRM-lacZ-Pcat-E1 | Simultaneous expression of MazE1-Leup on the basis of pPef1-IRM-lacZ | This study |
| pPdlt-lacZ | Expression of report gene lacZ controlled by promoter of dlt operon | This study |
| pPdlt-lacZ-Pcat-E1 | Simultaneous expression of MazE1-Leup on the basis of pPdlt-lacZ | This study |
| pACYC-his-E1 | Expression of MazE1-Leup with N-terminus His tag | This study |
| pACYC-his-E1-F1 | Expression of MazE1-Leup with N-terminus His tag and label free MazF1-Leup | This study |
| pUT18C-mazE1 | Fusion expression of MazE1 and T18 | This study |
| pKT25-mazF1 | Fusion expression of MazF1 and T25 | This study |
-表示相关因子缺失或相关蛋白功能丧失，Δ表示基因或DNA片段被敲除
- indicates loss of related factor or loss of function of related protein; Δ represents the knocked-out gene or DNA fragment;
1.1.2 主要试剂和仪器
高保真DNA聚合酶I-
水平电泳仪、垂直电泳仪、成像仪(荧光成像)，上海天能科技有限公司；PCR仪，Bio-Rad公司；摇床，上海智城分析仪器制造有限公司；培养箱，上海博迅实业有限公司医疗设备厂；酶联免疫分析仪，杭州奥盛仪器有限公司；台式高速冷冻离心机，湖南湘仪实验室仪器开发有限公司；分光光度计，Eppendorf公司；荧光定量PCR系统，ThermoFisher Scientific公司。
1.1.3 引物
本研究所用引物见
引物 Primers name | 序列 Primer sequences (5′→3′) |
|---|---|
| mazF1-f | GGGCTCGAGATGAGTGATGATATTCGCG |
| mazF1-r | CCCGGTACCTCATTCTAAATCAGATAAAAACGC |
| mazF2-f | GGGCTCGAGATGAGTTACAAGCCTAGGC |
| mazF2-r | CCCGGTACCTTAAACCCAACTGGCTGG |
| mazF3-f | GGGCTCGAGATGAGTGAATTAGATCCCAG |
| mazF3-r | CCCGGTACCTTATCCTTTGAAAGGTATCTTTC |
| mazF1-R28A-f | TGGCTCCGGCATTTGTAGTGAAATACG |
| mazF1-R28A-r | TACAAATGCCGGAGCCACCTTGGAGCCTACTTCTCGC |
| mazF1-T46A-f | AGCTAGTCAATATGATGATAAATCAGAATATTTTAAG |
| mazF1-T46A-r | CATCATATTGACTAGCTATTTTGTAATAAGTAATCCGCTGATTATC |
| mazF2-I50T-f | GCCAATAACTCACGGTGATTGGGAGTTTGC |
| mazF2-I50T-r | CACCGTGAGTTATTGGCGTCACAATGGCAA |
| mazF3-E18G&K20E-f | CCATATGGAGACGAAAGTGATTCAAAAACTAGACCTGCTTTA |
| mazF3-E18G&K20E-r | CTTTCGTCTCCATATGGTATGTTTACCACGTAAACTTTCA |
| HisE1OE-f | ACCATCATCACCACAGCCAGGATCCGATGAAAGCAATTCAAGTATCTGCTCG |
| HisE1OE-r | CTTAAGCATTATGCGGCCGCAAGCTTTCATTGCAAAAATTCCTCACGG |
| HisE1-F1-OE-f | GCCACGCGATCGCTGACGTCGGTACCATGAGTGATGATATTCGCGAGCA |
| HisE1-F1-OE-r | GCAGCGGTTTCTTTACCAGACTCGAGTCATTCTAAATCAGATAAAAACGCCA |
| F1-KT25-f | GGCGGGCTGCAGGGTCGACTCTAGAGATGAGTGATGATATTCGCGAGCA |
| F1-KT25-r | TACGCTGGATAGGTACCCGGGGATCCTCATTCTAAATCAGATAAAAACGCCA |
| E1-UT18C-f | AACGCCACTGCAGGTCGACTCTAGAGATGAAAGCAATTCAAGTATCTGCTCG |
| E1-UT18C-r | AATTCGAGCTCGGTACCCGGGGATCCTCATTGCAAAAATTCCTCACGG |
| EF1-prom-f | CCCGCGGCCGCGCTTTCTCTAGATAGCAAGC |
| EF1-prom-r | CCCGGATCCGCTTTCATAATGTGCACCC |
| EF1-prom-IRM-f | ACATATACGCCATAAATGTGTTATAATTTAAGTAAAG |
| EF1-prom-IRM-r | ACACATTTATGGCGTATATGTTATCTATTGTTAGTAATTATG |
| EF1-probe-FAM-r | GCTTTCATAATGTGCACCC |
| cat-prom-f | CACTGGTGATACCATTCGCGATGATCGGCACGTAAGAGGTTC |
| cat-prom-r | TTTAGCTTCCTTAGCTCCTGAAAAT |
| EF1-cat-prom-f | CAGGAGCTAAGGAAGCTAAAATGAAAGCAATTCAAGTATCTGCTC |
| EF1-cat-prom-r | TCGTCATCCGGAGGCTCGCGATCATTCTAAATCAGATAAAAACGCC |
| E1-cat-prom-r | TCGTCATCCGGAGGCTCGCGATCATTGCAAAAATTCCTCACGG |
| Pef1-3510D-f | TTAAGTAAAGTATATTAAGTGGGG |
| Pef1-3510D-r | ACTTAATATACTTTACTTAAGTTAGTAATTATGCTGAATTGG |
| 16SqF | CAATGGGCGAAAGCCTGATG |
| 16SqR | GCACGTATTTAGCCGTCCCT |
| E1qF | GCTCGTGTTGATCCAAGCAT |
| E1qR | GGCTGTTGATGGGTCTCTTGA |
| F1qF | CAAGGTGAGACCGGCATTTG |
| F1qR | GCCCGCATAAACCCAATCTT |
| dlt-acpS-f | ACCTTTCTTCGCCAAGGGTT |
| dlt-acpS-r | CCTGCCAATGGACTTTTTCACC |
| acpS-alr-f | AGTCCATTGGCAGGATATTGAA |
| acpS-alr-r | GTTGCAACAGCAAAGTCCGT |
| Pdlt-f | GAGACCGGTCGTTTTGCGGCCGCGTCGCTCCAATATTGAAACACAAT |
| Pdlt-r | ACGGGATCTATCATTGGATCCCCGCTGAAAAAACTGTTTTATCAT |
| Pdlt-probe-f-fam | GTCGCTCCAATATTGAAACAC |
| Pdlt-probe-r | CCGCTGAAAAAACTGTTTTATC |
(待续)
；下划线标记的核苷酸为限制性内切酶的识别位点。
The underlined nucleotides indicate the recognition sequences of restriction endonucleases.
1.2 重组质粒的构建
1.2.1 潜在毒素蛋白毒性验证质粒的构建
按照细菌基因组提取试剂盒说明书提取假肠膜明串珠菌L64的全基因组DNA。以该基因组DNA为模板，利用特异性引物mazF1-f/r、mazF2-f/r及mazF3-f/r (
1.2.2 毒素蛋白定点突变相关载体的构建
按照质粒抽提试剂盒说明书从大肠杆菌TOP10/pBAD-F1中提取重组质粒pBAD-F1。以提取的质粒pBAD-F1为模板，使用引物mazF1-R28A-f/r (
1.2.3 细菌双杂交质粒的构建
以假肠膜明串珠菌L64的基因组DNA为模板，利用引物对F1-KT25-f/r及E1-UT18C-f/r (
1.2.4 抗毒素MazE1-Leup及毒素抗毒素复合物MazEF1-Leup表达质粒的构建
以假肠膜明串珠菌L64的基因组DNA为模板，使用引物HisE1OE-f/r (
1.2.5 基于LacZ的相关报告质粒的构建
以假肠膜明串珠菌L64的基因组DNA为模板，使用引物EF1-prom-f/r (
1.3 检测毒素对宿主细胞的毒性及抗毒素对毒素的中和作用
将包含pBAD/myc-hisA或潜在毒素蛋白诱导表达质粒的大肠杆菌TOP10接种于含100 μg/mL氨苄青霉素的LB培养基，37 ℃、220 r/min振荡培养过夜。随后，以1%的接种量转接至新鲜的LB培养基，37 ℃培养至OD600值达到0.2-0.3。将培养物均分为2等份，分别加入0.2%葡萄糖及0.2%的l-阿拉伯糖进行诱导，然后37 ℃继续培养1 h。之后，进行梯度稀释(1
将包含pBAD-F1及pACYC-his-E1的大肠杆菌BL21(DE3)接种至含100 μg/mL氨苄青霉素及25 μg/mL氯霉素的LB培养基，37 ℃培养过夜。同样地，以1%的接种量转接至新鲜的LB培养基，37 ℃培养至OD600值达到0.2-0.3，将培养物均分成4份，分别加入0.2%葡萄糖、0.5 mmol/L的IPTG、0.2% l-阿拉伯糖及0.5 mmol/L的IPTG和0.2% l-阿拉伯糖，37 ℃继续培养，每隔半小时取样测定OD600，连续测定2 h。诱导1 h后，取样进行梯度稀释(1
1.4 抗毒素MazE1-Leup及毒素-抗毒素复合物MazEF1-Leup的表达与纯化
将表达质粒pACYC-his-E1及pACYC-his-E1-F1分别转化至大肠杆菌BL21(DE3)感受态细胞中，获得相应的表达菌株大肠杆菌BL21(DE3)/pACYC-his-E1及大肠杆菌BL21(DE3)/pACYC-his-E1-F1。以MazE1-Leup的表达与纯化为例进行阐述：将大肠杆菌BL21(DE3)/pACYC-his-E1接种于含25 μg/mL氯霉素的LB培养基中，于37 ℃、220 r/min振荡培养过夜。以1%的接种量转接至含25 μg/mL氯霉素的新鲜LB培养基， 37 ℃、200 r/min培养至OD600达到0.5-0.6之间，加入终浓度为0.5 mmol/L的IPTG诱导MazE1-Leup的表达。诱导结束后，4 ℃、8 000 r/min离心5 min收集菌体。用buffer A (25 mmol/L Tris-HCl pH 7.5、150 mmol/L NaCl)重悬菌体，并通过超声破碎菌体，将破碎后的菌体裂解液于4 ℃、12 000 r/min离心45 min，保留上清液。基于重组蛋白His6-MazE1-Leup中的His标签，利用Ni-NTA柱进行亲和层析纯化MazE1-Leup。利用buffer B (25 mmol/L Tris-HCl pH 7.5、150 mmol/L NaCl、50 mmol/L咪唑)洗涤杂蛋白，然后用buffer C (25 mmol/L Tris-HCl pH 7.5、150 mmol/L NaCl、500 mmol/L咪唑)洗脱，获得目标蛋白His6-MazE1-Leup。最终利用buffer D (25 mmol/L Tris-HCl pH 7.5、150 mmol/L NaCl、5%甘油)通过超滤对目标蛋白进行脱盐及浓缩。通过SDS-PAGE检测目标蛋白MazE1-Leup的表达情况与纯度。基于MazE1-Leup与MazF1-Leup的直接相互作用，利用MazE1-Leup携带的His标签来纯化MazEF1-Leup复合物。纯化过程与MazE1-Leup的纯化过程相同。最后，利用MALDI-TOF MS对纯化产物进行鉴定。
1.5 细菌双杂交(bacterial adenylate cyclase-based two-hybrid, BACTH)
将包含pKT25-mazF1及pUT18C-mazE1的大肠杆菌BTH101接种于含50 μg/mL卡那霉素及100 μg/mL氨苄青霉素的LB培养基中，30 ℃培养过夜。以1%的接种量转接至新鲜LB培养基，继续培养至OD600值达到0.1左右，加入终浓度为0.5 mmol/L的IPTG进行诱导，数小时后取样测定细胞内β-半乳糖苷酶的活性。同时，取100 μL培养物，4 ℃、8 000 r/min离心5 min收集菌体，用5 μL新鲜LB培养基重悬菌体后点样至含有X-gal的LA平板，30 ℃培养箱倒置培养过夜，观察颜色变化。采用同样的方法处理阳性对照大肠杆菌BTH101/pKT25-zipper/pUT18C-zipper及阴性对照大肠杆菌BTH101/pKT25/pUT18C。
1.6 凝胶迁移实验(electrophoretic mobility shift assay, EMSA)
参考Li
1.7 总RNA的提取、RT-PCR及RT-qPCR
将假肠膜明串珠菌L64的30 ℃过夜培养物接种至新鲜的d-乳酸发酵培养基。对照组在发酵培养基中加入55 g/L的CaCO3，以中和产生的乳酸并稳定培养基的pH；实验组则不添加CaCO3，其他成分保持一致。经过48 h发酵后，按照RNA抽提试剂盒的操作说明书抽提总RNA，并使用DNase I在37 ℃处理RNA，去除残留的基因组DNA。随后，利用逆转录试剂盒合成cDNA。设计特异性RT-qPCR引物(
2 结果与分析
2.1 假肠膜明串珠菌L64基因组编码3对潜在的MazEF类II型毒素-抗毒素系统
利用TADB 3.0在线工具TAfinder 2.
编号 Number | 毒素 Toxin | 抗毒素 Antitoxin | 毒素-抗毒素结构域 Toxin-antitoxin domain |
|---|---|---|---|
| mazEF1-Leup | OYT93_01690 | OYT93_01685 | MazF-RelB/DinJ |
| mazEF2-Leup | OYT93_03030 | OYT93_03025 | MazF-PemK |
| mazEF3-Leup | OYT93_04285 | OYT93_04280 | MazF-MazE |
2.2 毒素蛋白MazF1-Leup对大肠杆菌具有强烈毒性
构建潜在毒素蛋白过表达质粒并分别导入大肠杆菌TOP10细胞中，同时，将空白质粒pBAD/myc-hisA导入大肠杆菌TOP10细胞中作为对照。在抑制条件(添加0.2%葡萄糖)和诱导条件(添加0.2% l-阿拉伯糖)下分别生长1 h后，通过稀释点板实验检测毒素的表达对宿主生长的影响。在抑制条件下，毒素蛋白表达宿主与空白对照一样，均正常生长；在诱导条件下，大肠杆菌TOP10/pBAD-MazF1的生长受到明显抑制，而大肠杆菌TOP10/pBAD-MazF2和大肠杆菌TOP10/pBAD-MazF3与空白对照大肠杆菌TOP10/pBAD一样，均正常生长(
图1 假肠膜明串珠菌L64染色体编码的MazF毒素的毒性鉴定。A：包含不同毒素的重组菌株Escherichia coli TOP10的梯度稀释滴板实验。+/-分别表示添加/未添加0.2% l-阿拉伯糖。B：同源MazF毒素多序列比对。星号标记的是活性位点。C：包含不同突变毒素的重组菌株E. coli TOP10的梯度稀释滴板实验。
Figure 1 Toxicity assessment of MazF toxins encoded by Leuconstoc pseudomesenteroides L64 chromosome. A: The 10-fold dilutions of Escherichia coli TOP10 harbouring pBAD/myc-hisA constructs, as indicated, spotted onto LA plates. +/- denotes the presence or absence of 0.2% L-arabinose. B: Multi-sequence alignments among MazF toxins. Amino acid residues marked with asterisks indicated the active sites. C: The 10-fold dilutions of E. coli TOP10 harbouring pBAD/myc-hisA constructs, as indicated, spotted onto LA plates.
多序列比对结果显示，MazF2-Leup和MazF3-Leup的潜在活性位点均发生突变，具体来说，MazF2-Leup的Ile (50)取代了Thr，MazF3-Leup的Glu (18)和Lys (20)分别取代了Gly和Glu (
2.3 抗毒素MazE1-Leup可以中和MazF1-Leup的毒性
将MazE1-Leup过表达载体pACYC-His-E1与MazF1-Leup过表达载体pBAD-F1共同导入大肠杆菌BL21(DE3)感受态细胞中。作为对照，将pACYCDuet-1及pBAD/myc-hisA也导入大肠杆菌BL21(DE3)。在培养基中分别加入IPTG、阿拉伯糖或IPTG和阿拉伯糖，检测宿主生长曲线。加入IPTG诱导MazE1-Leup的表达后，宿主细胞与空白对照一样正常生长(图
图2 MazE1-Leup通过与MazF1-Leup直接相互作用中和MazF1-Leup毒性。A：重组菌株Escherichia coli TOP10生长曲线；B：重组菌株E. coli TOP10梯度稀释滴板实验；C：MazE1-Leup与MazF1-Leup细菌双杂交实验；D：His6-MazE1-Leup及复合物His6-MazE1-F1的纯化。
Figure 2 MazE1-Leup neutralized toxicity of MazF1-Leup through direct interaction. A: Growth curves of Escherichia coli TOP10 expressing MazE1-Leup, MazF1-Leup or MazEF1-Leup complex; B: The 10-fold dilutions of E. coli TOP10 harbouring pBAD/myc-hisA constructs, as indicated, spotted onto LA plates; C: Bacterial two hybrid experiment of MazE1-Leup and MazF1-Leup; D: Purification of His6-MazE1-Leup and His6-MazE1-F1 complex.
2.4 mazE1-Leup与mazF1-Leup形成操纵子
II型抗毒素与毒素基因形成操纵子结构，mazE1-Leup与mazF1-Leup之间存在4个碱基的重叠(
图3 mazE1-Leup与mazF1-Leup形成操纵子。A：mazEF1-Leup基因结构示意图；B：RT-PCR检测mazEF1-Leup的遗传结构(gDNA表示基因组DNA)。
Figure 3 mazE1-Leup and mazF1-Leup consist of an operon. A: Schematic representation of mazEF1-Leup genetic structure. B: Genetic organization of mazEF1-Leup confirmed by RT-PCR (gDNA denotes genomic DNA). M: DNA marker.
2.5 MazE1-Leup及MazEF1-Leup复合物均能抑制自身启动子的转录活性
利用在线工具BPROM，在mazEF1-Leup的上游非编码区识别到了一个潜在的启动子Pef1(
图4 MazE1-Leup通过与启动子结合抑制mazEF1-Leup系统的转录。A：在mazEF1-Leup上游识别到的潜在启动子；B：报告质粒示意图；C：基于lacZ的报告系统结果表明MazE1-Leup或MazEF1-Leup均能抑制mazEF1-Leup启动子的转录；D、E：EMSA检测MazE1-Leup (D)或MazEF1-Leup (E)与启动子Pef1之间的相互作用；F：通过在EMSA反应体系中添加特异性竞争探针检测MazE1-Leup与Pef1之间是否存在特异性相互作用。
Figure 4 MazE1-Leup inhibited transcription of mazEF1-Leup by binding to promoter. A: A putative promoter was identified upstream of mazEF1-Leup; B: Schematic diagram of reporter plasmids; C: Transcription of mazEF1-Leup inhibited by MazE1-Leup or MazEF1-Leup was confirmed by lacZ based reporter system; D, E: Interaction between Pef1 and MazE1-Leup (D) or MazEF1-Leup (E) was revealed with EMSA; F: Specific interaction between Pef1 and MazE1-Leup was further confirmed by adding specific competitor within EMSA reaction system.
已知的II型抗毒素通过与启动子中的反向重复序列(inverted repeat, IR)结合来抑制启动子的转录，在Pef1中识别到一个IR (TAACAaaatgTGTTA)，位于-35区和-10区之间，并且部分与-10区重叠(
图5 MazE1-Leup与mazEF1-Leup启动子中的IR序列(TAACAaaatgTGTTA)结合。A：Pef1中IR突变后的相关质粒示意图(用于检测IR突变后，MazE1-Leup是否还能抑制Pef1转录)；B：MazE1-Leup不能抑制IR突变后的Pef1转录；C：MazE1-Leup不能与IR突变后的Pef1相互作用。
Figure 5 MazE1-Leup binds to the IR (TAACAaaatgTGTTA) of mazEF1-Leup promoter. A: Schematic diagram of plasmids constructed for detection of effect of IR mutation on the transcription of Pef1; B: MazE1-Leup can’t inhibit transcription of Pef1 with IR mutation; C: MazE1-Leup can’t bind to the Pef1 with IR mutation.
2.6 酸压力诱导mazEF1-Leup的转录
假肠膜明串珠菌L64发酵合成乳酸时，随着乳酸的不断积累，培养基中的pH值会发生显著变化。在发酵48 h后，与对照组(即在培养基中加入55 g/L的CaCO3以调节并保持pH稳定)相比，实验组的pH从6.2降低至4.5。为了探究培养基中pH值的变化是否能诱导mazEF1-Leup的转录，利用RT-qPCR检测了发酵过程中mazEF1-Leup的mRNA水平变化。结果显示，与对照组相比，mazE1-Leup的mRNA水平升高了约4倍(
图6 MazE1-Leup抑制dlt-acpS-alr操纵子的转录。A：酸压力诱导mazE1-Leup及dltA的转录；B：dlt操纵子、acpS及alr基因示意图(方框标记的是dlt操纵子启动子的-35和-10区；IR表示dlt操纵子启动子发现的相似反向重复序列)；C：dlt基因与acpS及alr形成操纵子(M: DNA marker)；D：MazE1-Leup抑制dlt操纵子启动子的转录；E：MazE1-Leup与dlt-acpS-alr操纵子的启动子结合。
Figure 6 MazE1-Leup inhibited the transcription of dlt-acpS-alr operon. A: Transcription of mazE1-Leup and dltA was induced under acid stress condition; B: Schematic representation of dlt operon, acpS and alr gene locus (Putative promoter of dlt operon was indicated by -35 and -10 box; Inverted arrows pair denotes the similar IR); C: dlt gene cluster, along with acpS and alr forms an operon as determined by RT-PCR (M: DNA marker); D: Transcription activity of Pdlt was inhibited by MazE1-Leup; E: MazE1-Leup binds to the promoter of the dlt-acpS-alr operon.
2.7 MazE1-Leup抑制dlt-acpS-alr操纵子的转录
利用MEME中在线工具FIM
3 讨论与结论
本研究在假肠膜明串珠菌L64基因组中识别到了3对潜在的mazEF类TA系统，分别是OYT_01690-01685、OYT_03030-03025及OYT_04285-04280。根据以下实验结果确定mazEF1-Leup (OYT_01690-01685)编码一个真正的II型TA系统。(1) 在3个潜在的毒素蛋白中，仅MazF1-Leup (OYT_01690)能够抑制大肠杆菌的正常生长，而其保守活性位点的突变则导致其毒性丧失，mazE1-Leup (OYT_01685)编码的抗毒素蛋白，通过与MazF1-Leup直接相互作用，达到中和其毒性的目的；(2) mazE1-Leup与mazF1-Leup发生共转录，形成操纵子结构；(3) MazE1-Leup及MazEF1-Leup复合物能够抑制自身启动子的转录；(4) MazE1-Leup通过与启动子中的IR序列 (TAACAaaatgTGTTA)结合抑制自身转录。
II型TA系统作为全局调控元件正日益受到关注，其主要原因之一在于II型毒素蛋白利用RNA酶活性在全局范围内选择性切割mRNA，从而调控全局性蛋白翻译。除毒素外，越来越多的实验证据表明，同时包含与毒素结合和DNA结合的模块化结构的抗毒素，可能作为通用型转录调控因子参与细菌其他重要生理生化过程的调控。在本研究中，在dlt操纵子的启动子中发现了与IR (TAACAaaatgTGTTA)相似的回文序列TAACAtattgaaatatatgTGTTA。同时，RF-qPCR结果显示，随着生长过程中乳酸等有机酸的积累导致环境pH下降，会诱导mazEF1-Leup和dlt操纵子转录水平上升。因此，推测MazE1-Leup可能通过调控dlt操纵子的转录增强宿主的耐酸性。研究发现，dlt操纵子广泛分布于革兰氏阳性细菌，参与细胞壁中脂磷壁酸(lipo-teichoic acid, LTA)的d-丙氨酰化修饰，与乳酸菌的酸耐受性相
图7 抗毒素MazE1-Leup协助假肠膜明串珠菌L64应对酸压力的调控机制
Figure 7 Proposed regulatory mechanism of MazE1-Leup in assisting L. pseudomesenteroides L64 to cope with acid stress.
在dlt-acpS-alr操纵子的内部还识别到了一个与IR (TAACAaaatgTGTTA)相似的回文序列TAACAcgatTGTTA (
基因编号 Gene number | 编码的蛋白功能 Protein function | 回文序列 Palindromic sequence |
|---|---|---|
| OYT93_01685 | Antitoxin MazE1-Leup | TAACAaaatgTGTTA |
| OYT93_01215 | DltX | TAACAtattgaaatatatgTGTTA |
| OYT93_01245 | Alanine racemase Alr | TAACAcgatTGTTA |
| OYT93_00860 | NADH-dependent flavin oxidoreductase | TAACAtatcgTGTTA |
| OYT93_06375 | phage tail tube assembly chaperone | TAACAttcaaTGTTA |
| OYT93_07930 | Hypothetical protein | TAACAcTGTTA |
| OYT93_08975 | LysM and FRQ1 domains containing protein | TAACAtTGTTA |
作者贡献声明
李鹏：研究构思和设计、开展实验、处理数据、撰写和修改论文；刘兰：开展实验、处理数据、修改论文；章帅文：开展实验；王通：协助实验操作；黄筱萍：设计实验、修改论文。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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