摘要
目的
土壤微生物耐受单一重金属或有机污染物的生物调控机制已被广泛研究,但微生物应对复合污染的生物学机制仍不清楚。本研究旨在通过转录组学揭示粪肠球菌HHT-1耐受镉(Cd)和苯胺(aniline, AN)单一及复合污染胁迫的生物学应对机制。
方法
采用转录组学方法,探究在半数抑制浓度(IC50)下HHT-1在Cd (150 mg/L)和苯胺(2 g/L)单一及复合胁迫(Cd浓度150 mg/L+苯胺浓度2 g/L)的转录调控机制。
结果
在Cd单一胁迫下,HHT-1通过增强金属结合蛋白和转运蛋白基因的表达,促进C
结论
粪肠球菌HHT-1对Cd和苯胺复合胁迫的转录调控机制结合了单一Cd胁迫和单一苯胺胁迫下的转录调控机制,但与单一Cd胁迫下的机制更为接近。在复合胁迫下,HHT-1主要通过增强细胞壁合成、C
肠球菌属(Enterococcus)是一类广泛分布于自然环境和人体内的革兰氏阳性球菌,常见的肠球菌主要包括粪肠球菌(E. faecalis)和屎肠球菌(E. faecium
肠球菌不仅定殖于人体和动物体内,还广泛存在于多样的自然环境中,例如土壤、水体以及植被
Cd作为一种典型的重金属污染物,具有毒性强、半衰期长、环境迁移性强、生物易吸收且不可逆的特
与单一污染相比,目前对于微生物拮抗复合胁迫(尤其是针对重金属与有机污染物的复合胁迫)的机制仍不清楚。本研究以分离自土壤中的粪肠球菌HHT-1作为研究对象,该菌能够耐受高浓度Cd和苯胺胁迫。通过转录组学揭示粪肠球菌HHT-1应对Cd和苯胺单一及复合胁迫的生物学调控机制。了解这些机制有助于预测和评估耐药微生物在不同环境中的生态作用和潜在影响,深入理解环境复合污染带来的额外生态风险——即条件致病性以及耐药基因的传播。
1 材料与方法
1.1 主要试剂和仪器
胰蛋白胨和酵母提取物,OXOID公司;氯化钠(NaCl),生工生物工程(上海)股份公司;氯化镉(CdCl2) (纯度≥99%)和苯胺(纯度≥99%),上海麦克林生化科技股份有限公司;cDNA逆转录试剂盒,ThermoFisher Scientific公司;RNA提取试剂TRIzol Reagent,Invitrogen公司。
电泳仪,Agilent公司;基因测序仪Illumina NovaSeq 6000,Illumina公司;NanoDrop 2000分光光度计,Invitrogen公司
1.2 菌株的富集和培养
实验菌株粪肠球菌(Enterococcus faecalis) HHT-1由天津大学地质微生物学团队从中国长春市吉林农业大学长期农田试验站(43°49′N,125°23′E)的玉米地表层土壤(0-20 cm)中分离纯化得到。粪肠球菌HHT-1的培养使用LB培养基(g/L):胰蛋白胨10.0,酵母提取物5.0,氯化钠10.0;并在30 ℃、150 r/min的摇床中培养。
1.3 镉和苯胺对粪肠球菌HHT-1的半数抑制浓度及联合毒性效应分析
参照文献[
生物量抑制率=(各处理组的生物量减少量/对照组的生物量)×100% | (1) |
1.4 粪肠球菌HHT-1响应镉和苯胺单一及复合胁迫实验处理
实验共设置4个处理组,每个处理组设置3个重复。处理组包括:(1) 对照(CK),在LB培养基中加入纯净水;(2) 镉单一胁迫处理(Cd),将一定体积的CdCl2母液加入LB培养基中,使最终Cd浓度为150 mg/L;(3) 苯胺单一胁迫处理(AN),将一定体积的苯胺母液加入到LB培养基中使最终苯胺浓度为2 g/L;(4) Cd和苯胺复合胁迫处理(Mix),将一定体积的CdCl2和苯胺母液加入到LB培养基中最终Cd和苯胺浓度分别为150 mg/L和2 g/L。确保各处理组的培养体系体积一致。将粪肠球菌HHT-1按1%的接种量接种到含有不同处理的LB培养基的锥形瓶中,置于30 ℃、150 r/min的摇床中培养。在细菌对数生长后期(CK处理组OD600值为0.482;Cd处理组OD600值为0.241;AN处理组OD600值为0.241;Mix处理组OD600值为0.096),将各处理组以10 000 r/min离心5 min,分离得到菌体,置于-80 ℃冰箱保存,用于RNA提取。
1.5 RNA提取和纯化
使用RNA提取试剂从样品中提取总RNA,并去除基因组DNA。对所提取的RNA进行浓度和纯度检测,采用琼脂糖凝胶电泳检测RNA完整性,测定RIN值。高质量的RNA样本(OD260/OD280=1.8-2.2,OD260/OD230≥2.0,RIN≥6.5,28S:18S≥1.0,总量>10 μg)用于构建测序文库。RNA检测合格后,取5 μg的总RNA,按照标准操作手册中的方法去除rRNA。使用Fragmentation Buffer将mRNA随机打断成短片段。按照Illumina的标准进行cDNA合成、末端修复、A碱基添加和测序接头连接。筛选出200-300 bp的cDNA目标片段,随后使用Phusion DNA聚合酶(NEB公司)进行PCR扩增。PCR扩增体系(50 μL):2×PCR预混液,正、反向引物(浓度10 μmol/L)各2.5 μL,DNA模板50 ng,补充ddH2O至50 μL。PCR扩增条件:98 ℃预变性30 s;98 ℃变性10 s,54 ℃退火30 s,72 ℃延伸45 s,共15个循环;72 ℃延伸10 min。通过TBS380定量后,使用测序平台进行测序。以上过程由上海凌恩生物科技有限公司完成。
1.6 基因差异表达分析和功能富集
利用reads per kilobase per million mapped reads (RPKM)法计算基因表达量,以识别实验组和对照组间的差异表达基因(differentially expressed genes, DEGs),RPKM法能有效消除基因长度和测序量差异对基因表达计算的影响,计算得到的基因表达量可直接用于比较不同样品间的基因表达差异。差异基因的筛选标准为|log2 fold change|≥1且错误发现率(false discovery rate, FDR)≤0.05。通过基因本体数据库(gene ontology, GO)和京都基因和基因组百科全书数据库(Kyoto encyclopedia of genes and genomes, KEGG)进行注释分析和显著性富集分析,确定差异基因涉及的生物功能及代谢途径,进而分析粪肠球菌HHT-1在Cd和苯胺单一及复合胁迫下的响应机制。
1.7 实时荧光定量PCR (RT-qPCR)验证
为验证RNA-seq实验结果,选取10个相关差异表达基因进行RT-qPCR分析,基因名称及引物序列见
Gene name | Forward primer (5′→3′) | Reverse primer (5′→3′) | References |
---|---|---|---|
thiE | GCAAATACGGATCCTGCT | GTTGGCGTTGTGAGGAGA |
[ |
nanE | CAACAACTGCATACGCTC | TGGAAGGCCCAGATTATG |
[ |
recA | TGAAACTAGGTGATGGTA | CTTAGGATAACCGCCTAC |
[ |
secE | TTCGGACGCATCTGGCTG | TGAATGCCACGAACACCA |
[ |
recO | CGCATCGTGATCCTGCTC | AGACGATGTCCAGCGACC |
[ |
efp | CGCAGCGAGTACCAGTAC | CTGGTTCTCGAGCATGAA |
[ |
GAPDH | CATGACAATTCGGCATCG | ATGTTCTGGGCAGCACCT |
[ |
rpoB | TGTCCGCATTGATCGCAC | TCAAGCGTATTTCGCAGG |
[ |
rpmE | TCGATCGACTGACCATCG | GTCTAGCATCCTGACTAC |
[ |
murC | GTCATCGATCGATCCTAC | CTATCGAGCTATCGATTG |
[ |
2 结果与分析
2.1 粪肠球菌HHT-1拮抗镉和苯胺单一及复合胁迫的差异表达基因分析
转录组的主成分分析(principal component analysis, PCA)能从整体上反映各组之间的总体表达差异和组内样本之间的变异度大小。结果显示,主成分1 (PCA1)与主成分2 (PCA2)可分别解释样品基因表达总体差异的92.35%和4.87%,PCA1和PCA2共同解释了总体方差的97.22% (

图1 HHT-1响应Cd和苯胺单一及复合胁迫的转录水平主成分分析(PCA)。CK为对照组;Cd为单一Cd处理;AN为单一苯胺处理;Mix为Cd和苯胺复合处理。
Figure 1 Principal component analysis of the transcriptional level response of HHT-1 to single and combined stress of Cd and AN. CK represents the control treatment; Cd represents the single Cd treatment; AN represents the single AN treatment; Mix represents the combined treatment of Cd and AN.
将符合|log2 fold change|≥11且FDR≤0.05的基因鉴定为差异表达基因,结果如

图2 Cd和苯胺单一及复合胁迫下HHT-1差异表达基因分析。A:Cd胁迫下差异表达基因火山图;B:苯胺胁迫下差异表达基因火山图;C:Cd和苯胺复合胁迫下差异表达基因火山图;D:AN、Cd和Mix处理中上调差异表达基因韦恩图;E:AN、Cd和Mix处理中下调差异表达基因韦恩图。
Figure 2 Analysis of DEGs in HHT-1 under single and combined stress of Cd and AN. A: Volcano plot of differentially expressed genes under Cd stress; B: Volcano plot of differentially expressed genes under AN stress; C: Volcano plot of differentially expressed genes under combined Cd and AN stress; D: Venn diagram of upregulated differentially expressed genes in AN, Cd and Mix treatments; E: Venn diagram of down regulated differentially expressed genes in AN, Cd and Mix treatments.
将各处理组的差异表达基因进行韦恩图(Venn diagram)可视化分析。图
2.2 粪肠球菌HHT-1拮抗镉和苯胺单一及复合胁迫的差异表达基因GO富集分析
为进一步揭示这些差异表达基因的特定生物学功能,将不同处理下的差异表达基因在GO (gene ontology)数据库进行了显著性富集分析。GO富集分析根据基因功能将基因和基因产物分为分子功能(molecular function, MF)、生物过程(biological process, BP)和细胞组分(cellular composition, CC) 3
Cd处理组共获得31个GO功能注释,其中上调基因富集到26个GO terms,下调基因富集到5个GO terms。AN处理组共得到61个GO功能注释,上调基因富集到13个GO terms。Mix处理组共获得55个GO功能注释,上调基因富集到41个GO terms,下调基因富集到14个GO terms (
Item | Upregulated GO terms | Downregulated of GO terms | ||||||
---|---|---|---|---|---|---|---|---|
BP | CC | MF | All | BP | CC | MF | All | |
Cd | 18 | 5 | 3 | 26 | 4 | 0 | 1 | 5 |
AN | 6 | 1 | 6 | 13 | 32 | 13 | 3 | 48 |
Mix | 29 | 9 | 3 | 41 | 7 | 4 | 3 | 14 |
Cd处理组中,生物过程富集到了细胞大分子代谢(cellular macromolecule metabolic process)、基因表达(gene expression)、RNA修饰(RNA modification)、tRNA修饰(tRNA modification)、核糖体蛋白复合物组装(ribonucleoprotein complex assembly)、大分子代谢过程(macromolecule metabolic process)、细胞大分子生物合成过程(cellular macromolecule biosynthetic process);细胞组分中富集了核糖体蛋白复合物(ribonucleoprotein complex)、核糖体(ribosome)、细胞质(cytosol);分子功能中富集了RNA结合(RNA binding)、核糖体结构成分(structural constituent of ribosome) (

图3 HHT-1响应Cd和苯胺单一及复合胁迫的上调差异表达基因GO富集分析。A:Cd胁迫下上调差异表达基因GO富集分析;B:苯胺胁迫下上调差异表达基因GO富集分析;C:Cd和苯胺复合胁迫下上调差异表达基因GO富集分析。
Figure 3 GO enrichment analysis of upregulated DEGS in HHT-1 in response to single and combined stress of Cd and AN. A: GO enrichment analysis of upregulated DEGS in response to Cd stress; B: GO enrichment analysis of upregulated DEGS in response to AN stress; C: GO enrichment analysis of upregulated DEGS in response to Cd stress the combined stress of Cd and AN.
AN处理组中,生物过程中富集到了对非生物刺激的响应(response to abiotic stimulus)、细胞脂质代谢(cellular lipid metabolic process)、脂质代谢过程(lipid metabolic process)、脂质生物合成(lipid biosynthetic process);细胞组分中上调了ATP酶复合体(ATPase complex);分子功能中上调了核苷酸结合(nucleotide binding)、核苷酸磷酸结合(nucleoside phosphate binding)、蛋白质结合(protein binding)、小分子结合(small molecule binding) (
Mix处理组中,生物过程中上调了细胞大分子代谢过程(cellular macromolecule metabolic process)、细胞大分子生物合成过程(cellular macromolecule biosynthetic process)、大分子生物合成过程(macromolecule biosynthetic process)、基因表达(gene expression)、肽生物合成过程(peptide biosynthetic process);细胞组分中富集了非膜结构细胞器(non-membrane-bounded organelle)、细胞内非膜结构细胞器(intracellular non-membrane-bounded organelle)、核糖体(ribosome)、核糖核蛋白复合物(ribonucleoprotein complex)、核糖体亚基(ribosomal subunit);分子功能中富集了核糖体的结构成分(structural constituent of ribosome)、RNA结合(RNA binding)、结构分子活性(structural molecule activity) (
2.3 粪肠球菌HHT-1拮抗镉和苯胺单一及复合胁迫的差异表达基因KEGG富集分析
为了更深入地探讨Cd和苯胺单一及复合胁迫对HHT-1代谢途径的影响,本研究利用KEGG数据库对相关基因进行归类,并按照它们参与的代谢通路或生物学功能进行了系统性分析。将不同处理组的全部差异基因进行KEGG富集分析,结果如

图4 HHT-1响应Cd和苯胺单一及复合胁迫的差异表达基因KEGG富集分析。A:Cd胁迫下差异表达基因KEGG富集分析;B:苯胺胁迫下差异表达基因KEGG富集分析;C:Cd和苯胺复合胁迫下差异表达基因KEGG富集分析。
Figure 4 KEGG enrichment analysis of DEGs in HHT-1 in response to single and combined stress of Cd and AN. A: KEGG enrichment analysis of DEGs in response to Cd stress; B: KEGG enrichment analysis of DEGs in response to AN stress; C: KEGG enrichment analysis of DEGs in response to the combined stress of Cd and AN.
Cd处理组富集到的通路包括细胞运动性(cell motility)、翻译(translation)、核苷酸代谢(nucleotide metabolism)、脂质代谢(lipid metabolism)、糖类生物合成和代谢(glycan biosynthesis and metabolism)、外源生物降解和代谢(xenobiotics biodegradation and metabolism)、信号传导(signal transduction) (
AN处理组上调的差异基因富集到生物合成其他次级代谢产物(biosynthesis of other secondary metabolites)、碳水化合物代谢(carbohydrate metabolism)、膜转运(membrane transport)、糖类生物合成与代谢(glycan biosynthesis and metabolism)、翻译(translation) (
Mix处理组富集的通路则包括细胞运动性(cell motility)、信号传导(signal transduction)、翻译(translation)、复制和修复(replication and repair)、外源生物降解和代谢(xenobiotics biodegradation and metabolism) (
2.4 粪肠球菌HHT-1耐受镉和苯胺单一及复合胁迫的关键基因表达情况分析
2.4.1 细胞壁合成相关基因的转录调控
在Cd、AN和Mix处理下,HHT-1上调了与肽聚糖生物合成(peptidoglycan biosynthesis)、壁酸生物合成(teichoic acid biosynthesis)、萜类骨架生物合成(terpenoid backbone biosynthesis)等与细胞壁合成相关的途径。在肽聚糖合成途径中,Cd处理组和Mix处理组分别上调了20个和18个基因,而AN处理组仅上调了9个相关基因。在壁酸合成中,Cd处理组和Mix处理组分别上调了18个和15个基因,而AN处理组仅上调了11个相关基因。Cd、AN和Mix三个处理组在萜类骨架生物合成中上调基因数量相似(

图5 Cd和苯胺单一及复合胁迫下HHT-1关键基因的表达情况分析。A:细胞壁合成相关基因;B:核糖体合成相关基因;C:ABC转运蛋白相关基因。
Figure 5 Analysis of the expression of key genes in HHT-1 under single and combined stress of Cd and AN. A: Cell wall synthesis-related genes; B: Ribosome synthesis-related genes; C: ABC transporter-related genes.
2.4.2 核糖体合成相关基因的转录调控
核糖体主要负责细胞内的蛋白质合成,作为细胞中的“蛋白质工厂”,核糖体通过翻译mRNA (信使RNA)上的遗传信息,将氨基酸组装成蛋白质。核糖体的正常功能对于细菌的生长、代谢以及应对环境胁迫至关重要。Cd处理组和Mix处理组分别上调了41个和42个核糖体合成相关基因。AN处理组仅上调了4个相关基因,却下调了28个相关基因。这可能反映了HHT-1在应对不同类型胁迫时资源和能量的重新分配以及调控策略的调整(
2.4.3 ABC转运蛋白相关基因的转录调控
在细菌体内,ABC转运蛋白利用ATP水解产生的能量来实现物质的跨膜转运,参与细胞内的多种生理过程,包括营养物质的摄取、代谢产物的排出、维持细胞内外物质平衡等。转录组数据显示,Cd处理组和Mix处理组分别上调了45个和41个相关基因,AN处理组仅上调了9个基因,却下调了32个相关基因(
2.4.4 氧化应激相关基因的转录调控
细菌在接触高浓度的必需或非必需金属后,普遍会产生氧化应激反应,这是由于活性氧(ROS)生成增加或抗氧化防御能力下降造成

图6 HHT-1拮抗ROS的机制分析
Figure 6 Analysis of the mechanism of HHT-1 antagonizing ROS.
2.4.5 DNA损伤修复相关基因的转录调控
Cd和苯胺能够引发细胞内的氧化应激反应,导致细胞产生大量ROS,这些ROS与细胞内的大分子物质反应,会引起脂质过氧化、蛋白质氧化和DNA损
Gene name | log2 fold change | ||
---|---|---|---|
AN | Cd | Mix | |
ndk, NME | - | 2.04 | - |
nrdD | - | 3.34 | - |
guaA, GMPS | - | 2.38 | 2.02 |
gmk, GUK1 | - | 2.51 | 2.44 |
udk, UCK | - | 3.60 | 4.17 |
pdp | -1.08 | - | - |
nagD | - | 2.33 | 2.49 |
cmk | 2.21 | 3.89 | 3.99 |
yfkN | -1.28 | -2.01 | -2.48 |
-表示基因差异表达不显著(-1<log2 fold change<1)。
- means differentially expressed genes are not significant (-1<log2 fold change<1).
2.5 致病基因及耐药相关基因表达情况分析
为了评估环境胁迫对微生物生态毒性的影响,本研究对粪肠球菌的致病基因和耐药基因进行了深入分析。研究发现这些基因在不同处理组的表达情况存在显著差异。粪肠球菌致病基因efaA在Cd处理组和Mix处理组中分别上调了7.7倍和8.3倍,而在AN处理组中下调了1.7倍。黏附蛋白基因ace在AN处理组中上调了3.1倍,而在Cd和Mix处理组中上调了6.50-8.25倍。与粪肠球菌表面黏附、免疫逃避相关的蛋白基因cpsB和cpsA在Cd和Mix处理组中上调了2.73-3.42倍,在AN处理组中仅上调了1.51-1.73倍。
Cd和Mix处理对粪肠球菌耐药基因的表达也有显著影响。Cd和Mix处理显著上调了HHT-1多种抗生素耐受基因的表达(Cd:51个,Mix:48个),而AN处理对抗生素耐受基因的上调作用较弱(17个)。其中,抗生素外排基因mdeA在Cd和Mix处理组中分别上调了5.18倍和5.40倍,抗生素抗性基因fabI在Cd和Mix处理组中分别上调了3.68倍和2.35倍,抗生素抗性基因nfsA在AN、Cd和Mix处理组中分别上调了1.16倍、3.62倍和4.65倍。抗生素外排泵相关基因TaeA、patA、patB、efrA,抗生素抗性相关基因vanYB、vanSG、vanRG等只在Cd和Mix处理组中显著表达,在AN处理组中未显著表达(

图7 HHT-1致病及耐药基因表达情况分析
Figure 7 Analysis of the expression of pathogenic genes and drug-resistant genes in HHT-1.
2.6 差异表达基因的RT-qPCR验证
为了验证RNA-seq数据的可靠性,本研究从转录组测序结果中选取了10个相关基因进行RT-qPCR验证,包括细胞代谢基因thiE,DNA修复基因recO和recA,RNA合成基因rpoB,转运蛋白基因secE,糖酵解基因nanE和GAPDH,细胞壁合成基因murC,以及蛋白合成基因efp和rpmE。如

图8 差异表达基因的RT-qPCR验证
Figure 8 Validation of DEGs using RT-qPCR.
3 讨论与结论
3.1 粪肠球菌HHT-1对镉和苯胺单一及复合胁迫的耐受能力
环境微生物对Cd和苯胺的耐受浓度范围通常分别为0.01-10 mg/L和30-700 mg/L。例如,Cd对明亮发光杆菌(Photobacterium phosphoreum) T3S 15 min的IC50值为0.537 mg/
3.2 粪肠球菌HHT-1在半数抑制浓度下拮抗镉和苯胺单一及复合胁迫的机制
镉能够对细胞造成损伤,尤其是对细胞壁和细胞膜。为了抵御Cd的毒性,HHT-1首先通过增加肽聚糖和壁酸的合成来强化细胞壁结构,从而增强其对环境胁迫的耐受性。HHT-1还上调了与ZinT/AdcA家族金属结合蛋白相关的基因来增强对C
高浓度Cd会引起细胞内的氧化应激反应,虽然Cd不直接参与ROS的产
苯胺主要通过电子传递链在细胞内参与ROS的产生对细胞产生毒
虽然HHT-1不是苯胺降解菌,但在面对苯胺胁迫时,HHT-1上调表达了苯胺降解酶相关的基因,如邻苯二酚2,3-双加氧酶(由catE基因编码,上调了4.8倍),证明了HHT-1以间位降解途径参与苯胺的降
镉和苯胺复合污染物对粪肠球菌HHT-1的联合毒性体现为拮抗效应(1+1<2)。粪肠球菌HHT-1在Cd和苯胺复合胁迫下,HHT-1的响应机制结合了Cd和苯胺单一污染胁迫的响应特征。首先,HHT-1上调了参与肽聚糖和壁酸生物合成的相关基因,增强了细胞壁的合成能力,从而有效抵御了Cd对细胞的毒性作用;通过上调ABC转运蛋白相关基因将C
从转录调控机制来看,粪肠球菌HHT-1在复合污染下的生物学响应机制结合了单一Cd和单一苯胺胁迫转录调控机制的特点,并且与单一Cd胁迫下的转录调控机制更为相似。PCA分析图显示,Mix处理组和Cd处理组在PC1和PC2上具有极高的相似性。在差异表达基因的韦恩图中,Mix处理组和Cd处理组具有最多的重叠差异基因,且显著高于Mix处理与AN处理的重叠差异基因(上调:704 vs. 31)。这一发现表明HHT-1在响应Cd和苯胺复合胁迫的转录调控时,其过程与单一Cd胁迫下的转录调控过程具有较高的相似性。
3.3 半数抑制浓度下镉和苯胺单一及复合胁迫对粪肠球菌HHT-1致病性及耐药性的影响
镉和苯胺作为单一及复合污染物,对生态系统构成了严重的危害,其毒性效应复杂且多样。深入研究Cd和苯胺的生态毒性机制,不仅能够更全面地理解这些污染物的潜在危害,还能为制定有效的环境保护策略和人类健康保护措施提供科学依据。肠球菌通过水平基因转移机制,能够获得对多种抗生素的耐药
本研究也有相同的发现,efaA在粪肠球菌中编码一种特定的抗原EfaA,EfaA是一种心内膜炎相关的表面蛋白,对肠球菌的致病性发挥重要作
作者贡献声明
渠素敏:数据整理、数据分析、初稿撰写和文稿修改;陈钰璇:实验分析;朱翔宇:数据分析;王钺博:数据分析;管冬兴:数据分析;张坚超:概念构思、方法设计和文稿修改;滕辉:概念构思。
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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