微生物学报

Rank	Gene	log₂ fold change	Annotation
↑: Up; ↓: Down; −: Negative number.
1↑	tte1563	Infinity	Enzyme related to GTP cyclohydrolase Ⅰ
2↑	sRNA00086	6.574 1	Unknown
3↑	thiM	5.894 4	Hydroxyethylthiazole kinase, sugar kinase family
4↑	tte2506	4.981 7	Hypothetical protein
5↑	thiE	4.897 6	Thiamine monophosphate synthase
6↑	tte0230	4.650 1	Hypothetical protein
7↑	fabG5	4.535 2	Dehydrogenases with different specificities (related to short-chain alcohol dehydrogenases)
8↑	rpmI	4.176 2	Ribosomal protein L35
9↑	tte1519	3.996 0	Hypothetical protein
10↑	ptsN3	3.829 0	Phosphotransferase system mannitol/fructose-specific IIA domain (Ntr-type)
1↓	tte0239	−Infinity	Transposase
2↓	atpB	−Infinity	F0F1-type ATP synthase a subunit
3↓	atpE	−Infinity	F0F1-type ATP synthase c subunit/Archaeal/vacuolar-type H⁺-ATPase subunit K
4↓	atpF	−Infinity	F0F1-type ATP synthase b subunit
5↓	atpH	−Infinity	F0F1-type ATP synthase delta subunit
6↓	atpA	−Infinity	F0F1-type ATP synthase alpha subunit
7↓	atpG	−Infinity	F0F1-type ATP synthase gamma subunit
8↓	atpD	−Infinity	F0F1-type ATP synthase beta subunit
9↓	tte0854	−Infinity	Hypothetical protein
10↓	tte1171	−Infinity	Hransposase

Rank	Gene	log₂ fold change	Annotation
↑: Up; ↓: Down; −: Negative number.
1↑	tte1563	Infinity	Enzyme related to GTP cyclohydrolase Ⅰ
2↑	sRNA00086	6.574 1	Unknown
3↑	thiM	5.894 4	Hydroxyethylthiazole kinase, sugar kinase family
4↑	tte2506	4.981 7	Hypothetical protein
5↑	thiE	4.897 6	Thiamine monophosphate synthase
6↑	tte0230	4.650 1	Hypothetical protein
7↑	fabG5	4.535 2	Dehydrogenases with different specificities (related to short-chain alcohol dehydrogenases)
8↑	rpmI	4.176 2	Ribosomal protein L35
9↑	tte1519	3.996 0	Hypothetical protein
10↑	ptsN3	3.829 0	Phosphotransferase system mannitol/fructose-specific IIA domain (Ntr-type)
1↓	tte0239	−Infinity	Transposase
2↓	atpB	−Infinity	F0F1-type ATP synthase a subunit
3↓	atpE	−Infinity	F0F1-type ATP synthase c subunit/Archaeal/vacuolar-type H⁺-ATPase subunit K
4↓	atpF	−Infinity	F0F1-type ATP synthase b subunit
5↓	atpH	−Infinity	F0F1-type ATP synthase delta subunit
6↓	atpA	−Infinity	F0F1-type ATP synthase alpha subunit
7↓	atpG	−Infinity	F0F1-type ATP synthase gamma subunit
8↓	atpD	−Infinity	F0F1-type ATP synthase beta subunit
9↓	tte0854	−Infinity	Hypothetical protein
10↓	tte1171	−Infinity	Hransposase

基于转录组分析的腾冲嗜热厌氧杆菌thiD的热适应机制研究

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刘亚娟 ¹ , 郑航辉 ¹ , 刘原子 ¹ , 陈宜军 ² , 万学瑞 ¹ , 赵春林 ³ , 王川 ¹^,^* , 杨宇泽 ²^,^*

微生物学报 | 研究报告 2024,64(9): 3453-3473

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微生物学报 | 研究报告 2024, 64(9): 3453-3473

基于转录组分析的腾冲嗜热厌氧杆菌thiD的热适应机制研究

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刘亚娟¹, 郑航辉¹, 刘原子¹, 陈宜军², 万学瑞¹, 赵春林³, 王川¹^,^*, 杨宇泽²^,^*

作者信息

1 甘肃农业大学动物医学院, 甘肃兰州 730070

2 北京市畜牧总站, 北京 100101

3 天水师范学院, 甘肃天水 741000

RNA-seq reveals the role of thiD in the thermal adaptation of Thermoanaerobacter tengcongensis

Yajuan LIU¹, Hanghui ZHENG¹, Yuanzi LIU¹, Yijun CHEN², Xuerui WAN¹, Chunlin ZHAO³, Chuan WANG¹^,^*, Yuze YANG²^,^*

Affiliations

1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, Gansu, China

2 Beijing Municipal Animal Husbandry Station, Beijing 100101, China

3 Tianshui Normal University, Tianshui 741000, Gansu, China

出版时间: 2024-05-30 doi: 10.13343/j.cnki.wsxb.20240153

文章导航

摘要

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ThiD在腾冲嗜热厌氧杆菌中由thiD编码, 是硫铵素合成途径的关键酶。thiD的结构和功能已在真菌、酵母菌和植物中得到阐明。然而, thiD在嗜热生物中的功能仍不清楚。

【目的】探讨腾冲嗜热厌氧杆菌的嗜热机制并揭示thiD在不同温度下的功能及在热适应调控中的作用。【方法】利用同源重组技术构建腾冲嗜热厌氧杆菌的ΔthiD株, 观察并比较野生株和ΔthiD株在50、60、75和80 ℃下的生长趋势。通过转录组测序分析ΔthiD株与野生株在75 ℃的差异表达基因。通过实时荧光定量PCR分析比较野生株和ΔthiD株中13个基因和3个sRNAs在50、60、75、80 ℃下的转录水平。【结果】成功构建了ΔthiD株, 生长曲线结果显示其在50 ℃时, ΔthiD株生长速率与野生株无显著差异。然而, 在60 ℃和75 ℃时, ΔthiD株生长速度明显慢于野生株。另外, ΔthiD株在80 ℃时几乎不生长。转录组结果显示, 与野生株相比, ΔthiD株有503个差异表达基因, 其中包括278个上调差异表达基因和213个下调差异表达基因。KEGG分析表明, 以下途径与热适应有关, 包括硫胺素代谢、嘧啶代谢和嘌呤代谢、肽聚糖生物合成、脂肪酸代谢途径、氨基酸代谢途径、双组分系统、DNA复制、同源重组、错配修复、磷酸转移酶系统。通过实时荧光定量PCR分析发现, 在野生株和ΔthiD株中与嗜热机制相关的13个基因和3个sRNAs在特定温度下的转录水平发生了变化。【结论】thiD在腾冲嗜热厌氧杆菌热适应过程中发挥重要作用。本研究为揭示thiD在不同温度下的功能和在热适应过程中的调控机制提供实验数据和理论依据。

关键词

腾冲嗜热厌氧杆菌 / thiD / 转录组 / 差异表达基因 / 热适应

Abstract

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ThiD encoded by thiD in Thermoanaerobacter tengcongensis is a key enzyme in the biosynthesis of thiamine. The structure and functions of thiD have been elucidated in fungi, yeasts, and plants, while the role of thiD in thermophiles remains unclear.[Objective] This study aims to explore the thermal adaptation mechanism of T. tengcongensis and reveal the role of thiD in the thermal adaptation of T. tengcongensis at different temperatures. [Methods] The thiD-deleted mutant (ΔthiD) of T. tengcongensis was constructed by homologous recombination. The growth trends of the wild type (WT) and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were observed and compared. The differentially expressed genes (DEGs) between ΔthiD and WT cultured at 75 ℃ were determined by RNA-seq. The transcript levels of 13 genes and 3 sRNAs in WT and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were compared and analyzed by real-time PCR. [Results] ΔthiD was successfully constructed, with the growth rate not significantly different from WT at 50 ℃. However, ΔthiD show cased slower growth than WT at 60 ℃ and 75 ℃ and did not grow at 80 ℃. The transcriptome results revealed 503 DEGs in ΔthiD compared with WT, including 278 DEGs with up regulated expression and 213 DEGs with down regulated expression. The Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated the following pathways associated with thermophilic adaptation, involving thiamine metabolism, pyrimidine metabolism and purine metabolism, peptidoglycan biosynthesis, fatty acid metabolism, amino acid metabolism, two-component system, DNA replication, homologous recombination, mismatch repair, and phosphotransferase system. The transcript levels of 13 genes and 3 sRNAs related to thermal adaptation in WT and ΔthiD changed at specific temperatures. [Conclusion] thiD plays an important role in the thermal adaptation of T. tengcongensis. This study provides experimental data and a theoretical basis for revealing the role of thiD in the thermal adaptation of thermophiles at different temperatures.

Key words

Thermoanaerobacter tengcongensis / thiD / transcriptome / differentially expressed genes (DEGs) / thermal adaptation

引用本文

刘亚娟, 郑航辉, 刘原子, 陈宜军, 万学瑞, 赵春林, 王川, 杨宇泽. 基于转录组分析的腾冲嗜热厌氧杆菌thiD的热适应机制研究. 微生物学报, 2024 , 64 (9) : 3453 -3473 . DOI: 10.13343/j.cnki.wsxb.20240153

Yajuan LIU, Hanghui ZHENG, Yuanzi LIU, Yijun CHEN, Xuerui WAN, Chunlin ZHAO, Chuan WANG, Yuze YANG. RNA-seq reveals the role of thiD in the thermal adaptation of Thermoanaerobacter tengcongensis[J]. Acta Microbiologica Sinica, 2024 , 64 (9) : 3453 -3473 . DOI: 10.13343/j.cnki.wsxb.20240153

正文

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Thermophiles are heat-loving microorganisms that thrive in high temperatures above 50 ℃^[1]. Numerous attempts have been made to understand the mechanisms of thermal adaptation^[2]. However, thermal adaptation is a very complicated process that involves multiple factors. The completion of genome sequences of thermophiles has provided valuable data into the identification of crucial genes involved in thermal adaptation over time^[3]. Numerous studies indicated that factors such as the G+C content of the genome and mRNA^[4-5], uracil content in 16S rRNA gene^[6-8], tRNA modifications^[9-11], biosynthesis of fatty acids for energy metabolism^[12-13], amino acid composition and utilization^[14], as well as thermal adaptation proteins were all associated with thermal adaptation^[15]. Furthermore, various temperature- dependent proteins also played important roles in thermal adaptation^[16-17].

Thermoanaerobacter tengcongensis was a thermophilic and anaerobic bacterium that was isolated from a hot spring in Tengchong, Yunnan, China. This bacterium could grow from 50 ℃ to 80 ℃, with the optimum growth temperature at 75 ℃^[18]. Notably, the genome sequencing and annotation of T. tengcongensis were firstly completed in China^[3]. The proteomics and genomics related to the thermal adaptation of T. tengcongensis have also been studied^[19-20]. Genomic analysis showed that a significant proportion (86.7%) of the genes in T. tengcongensis were encoded on the leading strand of DNA replication. A strong correlation has been observed between the G+C content of tDNA and rDNA genes and the optimal growth temperature among sequenced thermophiles^[3]. Additionally, in the genome of T. tengcongensis, glucokinases exhibited thermal stability regulated by their interaction with HSP60^[21], while the ribosome recycling factor (tteRRF) demonstrated high thermophilic stability^[22]. Transcriptomic analysis has revealed over 1 200 differentially expressed genes (DEGs) in T. tengcongensis in response to cold shock, indicating the involvement in various biological processes such as cell wall and membrane remodeling, flagellar assembly, and sporulation^[23]. Furthermore, two CRISPR-Cas systems were identified in T. tengcongensis that responded to the changes in temperature^[24]. The proteomics analysis identified 251 out of 1 589 differentially expressed proteins that exhibited temperature-dependent expression in T. tengcongensis at 55, 65, 75 and 80 ℃ using isobaric tags for relative and absolute quantitation^[16]. Additionally, 251 temperature-dependent proteins were identified in T. tengcongensis by two-dimensional gel electrophoresis and matter-assisted laser desorption/ ionization time-of-flight mass spectrometry at 55, 75, and 80 ℃^[25]. T. tengcongensis had a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins^[26]. Despite doing these studies, the thermal adaptation mechanisms of T. tengcongensis remain unclear.

ThiD is a bifunctional hydroxymethylpyrimidine or methylpyrimidine phosphate kinase, which is a key enzyme in the synthesis of thiamine^[27]. Its function is to catalyze the phosphorylation of hydroxymethylpyrimidine (HMP) and hydroxymethylpyrimidine phosphate (HMP-P)^[27]. The biochemistry and structure of ThiD have been well studied in most bacteria, yeasts, and plants^[28-32]. However, the biological function of ThiD in thermophiles remain unknown. Therefore, in this study, the disruption of thiD was constructed to obtain a thiD disrupted mutant (ΔthiD) and subsequently its role related to the thermal adaptation T. tengcongensis was studied. The investigations would deepen our understanding of the thermophilic mechanism of T. tengcongensis.

1 Materials and Methods

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1.1 Strains and plasmids

T. tengcongensis MB4 (China General Microbiological Culture Collection Centre, deposit number CGMCC 1.2430) was grown in Tris-taurine- EDTA (TTE) medium as described previously^[20]. Escherichia coli DH5α was cultured in LB medium as described previously^[33]. pBOL01 is a shuttle plasmid between T. tengcongensise and E. coli^[20]. pBOL01:: ΔthiD is a thiD disruption plasmid.

1.2 Construction and identification of thiD disruption strain

The whole genome of T. tengcongensis was isolated by CTAB, and subsequently it was used as a template for PCR amplification of thiD left-arm and right-arm fragments with P1 and P2 primers (Table S1). The left arm of thiD and plasmid pBOL01 were double digested with Xho Ⅰ and Hind Ⅲ, and then ligated by T4 DNA ligase and transformed to obtain the recombinant plasmid pBOL01::thiD left arm; the left arm of pBOL01::thiD recombinant plasmid and the right arm of thiD were double enzyme digested with BamH Ⅰ and Xba Ⅰ, and then the disruption plasmid pBOL01:: ΔthiD was obtained by ligation and transformation. According to the description of Liu et al^[20], 0.1 mL of WT solution was combined with 10 mL of TTE medium at a temperature of 75 ℃ for a duration of 5 hours. Subsequently, 1 mL of this mixture was introduced into another TTE medium along with 50 µL of pBOL01:: ΔthiD and incubated at 60 ℃ for 3 hours. Following the liquefaction of TTE solid medium and heating to 70 ℃, the above bacterial solution was incorporated into the molten TTE solid medium. This was followed by the addition of 0.5 mL 100 µg/mL kanamycin, thorough mixing, and the transfer of the mixture into anaerobic tubes, which were then flattened and subjected to cultivation at 60 ℃ for a period of 3 to 4 days. Following a single colony grown from the wall of solid anaerobic tubes, individual colonies of ΔthiD were selected and propagated in TTE liquid medium supplemented with 0.5 mL of 100 µg/mL kanamycin. The cultures were maintained at a temperature of 60 ℃ for a period of 2 to 3 days until the liquid medium became turbid. Subsequently, the genome was isolated and verified by PCR using primer P3 (Table S1).

1.3 Determination of growth curves of WT and ΔthiD

In order to assess the growth of WT and ΔthiD, WT and ΔthiD were grown respectively at 75 ℃ in 100 mL of TTE medium as described previously^[20]. Once the optical density at 600 nm (OD₆₀₀) of the culture reached 0.6, 1 mL of the culture was transferred to a separate flask containing 100 mL of TTE medium. Subsequently, the culture values at OD₆₀₀ were determined every two hours at 50, 60, 75, and 80 ℃, respectively.

1.4 Total RNA preparation

The WT and ΔthiD strains were cultivated in TTE medium at 75 ℃ until an optical density of 0.8 at OD₆₀₀ was achieved. Subsequently, the cells were then collected through centrifugation, and total RNAs were isolated using TRIzol reagent (CWBIO, China). The extracted RNA was further purified with the RNeasy Mini Kit (QIAGEN) and treated with DNase Ⅰ (RNase-free) (Amboina) to eliminate any potential DNA contamination. The integrity and degradation of the treated RNA were initially assessed using 1% agarose gel electrophoresis. Subsequently, the purity of the RNA was determined using a NanoPhotometer^® spectrophotometer, and the concentration was measured using the Qubit^® RNA detection kit in a Qubit^® 2.0 Fluorimeter. Finally, the RNA integrity was precisely evaluated using the Agilent Technologies 2100 Bio analyzer's RNA Nano 6000 detection kit. The samples with a RIN value above 6 and a concentration exceeding 50 ng/μL were considered to have satisfactory quality.

1.5 RNA-sequencing and analysis of DEGs

Total RNA was used as input material for the RNA sample preparations. A sequencing library was generated using Illumina^®'s (NEB) NEBNext^®Ultra^TM Directional RNA Library Preparation Kit and an index code for the attribute sequence was added to each samples^[34]. The library construction was performed following the reference reported by Liu et al.^[23]. RNA sequencing was performed by Illumina NovaSeq 6000. Qualified sequences were mapped to the T. tengcongensis genome using Bowtie 2-2.2.3 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.3/) and paired-end clean reads were aligned to the reference genome using HTSeq v0.6.1^[35-36]. The fragments per kilobase of exon model per million mapped fragments (FPKM) of each gene was calculated using the featureCounts v1.5.0-p3 within the subread software for quantitative assessment^[37]. The differential expression analysis of two conditions with three biological replicates per condition, was performed using the DESeq2 R package (1.18.0)^[38]. The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate^[39]. The P value < 0.05 and |log₂ fold change| value > 1 were set as the threshold for significantly differential expression^[40].

The gene ontology (GO) enrichment analysis of DEGs was implemented by the cluster Profiler R package (3.8.1), and gene length bias was corrected^[41]. KOBAS was used to test the statistical enrichment of DEGs in KEGG pathways^[42]. The Diamond software (0.9.13) was utilized to compare the target gene sequences with the selected reference^[43], and then the network was established based on the known interaction of the selected reference species. The protein interactions of 141 DGEs in 30 KEGG pathways were analyzed by STRING (https://www.string-db.org/). Cytoscape (3.9.1) (https://Cytoscape.org/) was used to screen out central DEGs to the protein-protein interaction (PPI) networks^[44]. In the PPI networks, the FPKM of DEGs with a selection degree greater than 10 was used as the expression level to generate heatmaps. The FPKM value of differential genes of different strains was taken as the expression level to do hierarchical cluster analysis. Different colors represent different clustering information. Genes within the same group exhibited similar expression patterns, suggesting potential functional similarities or involvement in the same biological process. The lg (FPKM+1) values were normalized and clustered, with red indicating high-expression genes and blue indicating low-expression genes.

1.6 Real-time PCR

cDNA was synthesized from each RNA sample using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix (Transgen Biotech Co., Ltd, Beijing). The primers used for real-time PCR are listed in Table S1. The relative expression values of WT in thiD were assigned as 1 with the 16S rRNA gene used as a reference. All samples were analyzed in triplicate, and the data were processed using Light Cycler^® 96 software (version 1.1.0.1320), each reaction volume was 20 μL, and the relative expression values of the genes were calculated using the 2^−ΔΔCt method and the normalization method. The results were represented as means ± standard errors (SD)^[23]. The expression levels of 12 genes and 3 sRNAs associated with the thermal adaptation mechanism in the ΔthiD were assessed at temperatures of 50 ℃, 60 ℃, and 75 ℃ using real-time PCR. GraphPad Prism 8 software was employed for data analysis.

2 Results

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2.1 Construction and identification of thiD disruption strain

The PCR analysis demonstrated that both 1 108 bp thiD right arm and left arm were obtained (Figure 1A). The recombinant plasmid pBOL01::thiD left arm was successfully constructed (Figure 1B). By double enzyme digestion of recombinant plasmids pBOL01:: ΔthiD, 5 000 bp and 1 108 bp fragments were obtained (Figure 1C). The genome extracted from the ΔthiD strain was used as a template to amplify and obtained a 3 200 bp fragment, and the genome of the WT strain was used as a template to amplify and obtained a 2 800 bp fragment (Figure 1D). This indicated that the thiD disruption strain was successfully constructed.

2.2 Determination of growth curves of WT and ΔthiD

The growth rate of ΔthiD was not significantly different from that of the wild type (WT) strain at 50 ℃ (Figure 2A). Nevertheless, the growth rate of ΔthiD was significantly slower in comparison with the WT strain at 60 ℃ and 75 ℃ (Figure 2B, 2C). Obviously, ΔthiD almost did not grow at 80 ℃ (Figure 2D).

2.3 Transcriptome analysis of WT and ΔthiD strains

In this study, we selected differentially expressed genes through two levels of multiple of difference (|log₂ fold change| > 1) and significance level (P < 0.05) by DESeq2. By comparing the gene expression of the ΔthiD strain at 75 ℃ with that of the WT strain, a total of 503 DEGs were identified, by which consisted of 278 up regulated DEGs and 213 down regulated DEGs (Figure 3A, Table S2).

The top 10 up regulated and down regulated genes are listed in Table 1. Among the most up regulated genes, tte1563 encoded the enzyme related to GTP cyclohydrolase Ⅰ and thiM was a gene encoding hydroxyethylthiazole kinase.

The GO analysis revealed that there were 1 086 up regulated GO terms and 1 251 down regulated GO terms. The specific details are provided in table S3. The top 25 enrichment groups were evaluated based on their P values within the GO categories of biological process, cellular component, and molecular function, as depicted in Figure 3B. Noteworthy subcategories included carbohydrate derivative metabolic process, nucleobase-containing small molecule metabolic process, and nucleoside phosphate metabolic process. Nucleotide metabolic process, tetrapyrrole metabolic process, and ribose phosphate metabolic process were prominently represented. These results indicated that the DEGs of these processes were all involved in the process of thermal adaptation.

The analysis of enriched KEGG revealed that there were 96 pathways, with specific details provided in Table S4. Among these pathways, 30 important pathways related to thermal adaptation were further screened out. As shown in Figure 3C, the metabolic pathway was the largest number of annotation pathway. Additionally, the TCS, ribosome, 2-oxo-carboxylic acid metabolism, PTS, degradation of valine, leucine and isoleucine, homologous recombination, and mismatch repair pathways were related to thermal adaptation.

Furthermore, the PPI networks were constructed using 141 DEGs derived from 30 signaling pathways, including up regulated 77 DEGs and 64 down regulated DEGs. The protein interactions of 141 DGEs in 30 KEGG pathways were analyzed using STRING. According to Cytoscape program, 91 interacting proteins of DEGs in the PPI networks were identified. The key DEGs ranked by gene degree included tal, pgi, atpF, atpH, aroK, aroB, ANT_16660, ANT_13390, atpA, dnaN, and nuoG (Figure 3D). Subsequently, DEGs with a degree greater than 10 were selected, and their FPKM values were used to generate heatmaps. The heatmap consisted of 36 DEGs (19 up regulated and 17 down regulated DEGs) (Figure 3E). The pgi was involved in carbon metabolism pathway, while atpF and atpH were associated with in oxidative phosphorylation pathway. Additionally, the aroK and atpA participated in metabolic pathways, and DNA polymerase Ⅲ beta subunit (dnaN) participated in the citrate cycle.

To thoroughly examine the expression of genes identified in the RNA-seq analysis, we performed real-time PCR on three of the most highly expressed genes (thiE, tte0003, and napF3), three of the most up regulated genes (tte2227, ccmA2, and rnhA), two of the most down regulated genes (tte0272 and thiD), and two moderately repressed genes (galU and tte2411). The results from the real-time PCR were basically consistent with the transcriptome data, indicating that the transcriptome sequencing results had high reliability and could be used as a reference for bioinformatics (Figure 3F).

2.4 The expression of specific DEGs in ΔthiD at different temperatures

Using the real-time PCR, ΔthiD was compared with WT at 50, 60, 75 and 80 ℃ and the specific genes mentioned below were obtained. The expression of tte2227 at 50 ℃ was significantly lower than that of WT, while significantly higher at 60 ℃ (Figure 4B−4C). The thiE identified as a thiamine phosphate synthetase gene displayed higher expression levels than WT at 50, 60, 75 and 80 ℃, with the highest expression observed at 80 ℃ (Figure 4A−4D). In contrast, the thiD, a gene encoding a bifunctional hydroxymethylpyrimidine or methylpyrimidine phosphokinase, showed no expression at 50, 60, and 75 ℃ in comparison with that of WT, but exhibited increased expression at 80 ℃ when compared with that at 50 ℃ (Figure 4A−4D). Furthermore, the tte0620 was basically not expressed at 50 ℃ and 60 ℃, but was higher than WT at 75 ℃ (Figure 4B 4D). Similarly, the tte0003 was not expressed at 50 ℃, but was significantly higher than WT at 60 ℃ and 75 ℃ (Figure 4B−4D). Conversely, the expression levels of tte0272, rnhA and napF3 were significantly lower than those of WT at 50 ℃, but increased at 60 ℃ and 75 ℃ (Figure 4B−4D). On the other hand, only the ccmA2 and tte2763 exhibited significantly higher expression levels than WT at 60 ℃, while the ccmA2, tte2763, and tte2411 displayed significantly higher expression levels than WT at 75 ℃ (Figure 4C−4D). Furthermore, the tte2411 was not expressed at 50 ℃ and 60 ℃, but was significantly higher than WT at 75 ℃ (Figure 4B−4D). In addition, non-coding RNAs such as sRNA49, sRNA103, and sRNA104 exhibited different expression levels compared to WT at specific temperatures.

3 Discussion and Conclusions

收起

In this study, the thiD disrupted mutant did not grow at 80 ℃, and the growth rate at 50, 60 and 75 ℃ was slower than that of the WT strain, indicating that thiD played a role in the normal growth, metabolism, and survival of T. tengcongensis. Based on the transcriptome analysis, we suggested that high-temperature growth of T. tengcongensis was mainly associated with the expression of genes related to thiamine biosynthesis pathways, purine and pyrimidine metabolism, peptidoglycan biosynthesis, fatty acid biosynthesis, amino acid metabolism, TCS, DNA replication, recombination and repair, the ribosomal pathway, PTS, the central carbon metabolism, glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, and glycolysis.

3.1 Biological roles of thiD and thiE

Thiamine pyrophosphateas a cofactor for several enzymes were essential for the metabolism of carbohydrates and amino acids^[27]. In bacterial genomes, genes encoding enzymes were involved in thiamine biosynthesis pathways, such as the thiD, thiE, and thiM, as the key genes in thiamine metabolism and 2-oxo carboxylic acid metabolism. In T. tengcongensis, the thiD regulated the thiamine metabolic pathway by encoding a dipyromethylpyrimidine phosphokinase and catalyzing the phosphorylation of HMP and HMP-P to adapt to high temperature^[28]. And the expression levels of thiD at 50 ℃ and 60 ℃ and 75 ℃ were consistent with the trend of the analysis at different temperatures by Wang et al.^[24]. Other genes involved in thiamine metabolism included the thiE, which was the thiamine phosphate synthetase gene. And the deletion of thiD resulted in the increased expression and up-regulation of other genes involved in thiamine metabolism including the thiE, thiM, thi80, and nifS2, which ensured the stable growth of ΔthiD at different temperatures.

3.2 DEGs involved in purine metabolism and pyrimidine metabolism

The involvement of DGEs in purine and pyrimidine metabolism suggested that the key to maintaining thermophilic stability of T. tengcongensis at extreme growth temperatures was fundamentally dependent on the stability of genetic material. The high content and frequency distribution of guanine and cytosine in genetic material significantly enhanced the stability of proteins^[45]. In this study, purine metabolism demonstrated the upregulation of eight DEGs (nrdA, dnaN, purB, dnaE, purC, purM, purN, and purH) and pyrimidine metabolism showed the upregulation of five DEGs (dnaN, tmk, dut, nrdA, and dnaE). The results suggested that, in terms of nucleotide composition, the relative abundance of purines, particularly the increased frequency of adenine, in the coding sequences of thermophiles might contribute to genetic material stability by stabilizing the tertiary structure^[9]. In addition, other mechanisms such as modifications of rRNA, tRNAs, selective translation of codon-biased transcripts, and regulation of translation elongation were also considered as the positive ways to respond to environmental changes^[46-49].

3.3 DEGs involved in cell wall and membrane structures

Peptidoglycan biosynthesis was a crucial process in the formation of bacterial cell walls, serving as the main structural component and provided protection against environmental stress in various organisms^[50]. In this study, the expression of pgi encoding glucose-6-phosphate isomerase, which is involved in glycolysis, fructose-6-phosphate and UDP-N-acetylglucosamine biosynthesis, was up regulated. UDP-N- acetylglucosamine was an essential component and precursor of bacterial peptidoglycan^[51-52]. The up-regulation of the pgi implied that these pathways provided essential energy and precursor molecules for the metabolism of T. tengcongensis. Furthermore, the enhanced biosynthesis and accumulation of peptidoglycan were responsible for protecting the cell integrity of T. tengcongensis against temperature fluctuations.

Moreover, the fatty acid biosynthesis pathway could modulate the response of the cell membrane of thermophiles to different temperatures by altering branched-chain fatty acid content and controlling the biophysical properties of membrane phospholipids enabling them to survive in extreme environments^[12]. The results indicated that fatty acid and phospholipid biosynthesis enzyme phosphoacyltransferase (plsX) were involved in the formation of fatty acid and phospholipid, indicating that T. tengcongensis modified the original phospholipid structure during the process of thermal adaptation^[50]. This process involved precise adjustments to the composition of membrane lipids to maintain the necessary fluidity of cell membranes at different temperatures.

3.4 DEGs involved in amino acid metabolic pathways

Regulating the metabolism of amino acids has been identified as a potentially effective strategy for protecting cells from the adverse effects of temperature variations^[53]. This study revealed that the expressions of lysine, glutamate, valine, isoleucine, and tyrosine were up regulated, whereas alanine, histidine, glutamine, and threonine, were down regulated. By regulating the metabolic pathway of arginine and proline, and upregulating the degradation pathway of lysine, it would be possible to enhance the production of thermal adaptive proteins^[54]. This study indicated that significant alterations in the levels of these amino acids played a role in enhancing the thermal stability of proteins.

3.5 DEGs involved in two-component regulation

A two-component system was regarded as a common signaling pathway in many bacteria, serving as information processing pathways that linked external stimuli with adaptive responses within cells. In prokaryotes, signal transduction was mainly carried out by a two-component regulatory system^[55-56]. The two-component system was responsible for cellular responses to diverse environmental stimuli, effectively regulating gene expression^[57]. In this study, the up regulated cheY2 expression was consistent with the trend of RNA-sequencing analysis of cold shock response in T. tengcongensis, a bacterium harboring a single cold shock protein encoding gene reported by Liu et al.^[23]. In addition, the expression patterns of cheY7 at 80 ℃, as well as the baeS12 and ompR4 at 50 ℃ in T. tengcongensis, was consistent with the results found by Wang et al.^[24]. T. tengcongensis had a typical two-component system consists of two key elements: histidine kinases sensory transduction (ddpX and baeS12), which are sensitive to a specific environmental factor, and a response regulator ompR4, facilitating the transmission of the sensor signals and adapting the proper responses via the regulation of gene expression. These components play a key role in two typical two-component systems that transmit sensor signals and modulate gene expression to elicit suitable responses^[58]. This study indicates that the signal DEGs associated with this pathway in signal transduction plays a significant role in participating in the thermal adaptation mechanism of T. tengcongensis.

3.6 DEGs involved in DNA replication, recombination and repair

Three interconnected biological processes, specifically DNA replication, recombination, and repair, were essential for maintaining the fidelity and integrity of the genome^[59]. This study focused on analyzing the expression of DEGs associated with thermal adaptation of T. tengcongensis in DNA replication, mismatch repair and homologous recombination. Among them, the dnaE, dnaN and holB participated in low temperature induction. Additionally, the rnhB was associated with oriC-dependent chromosome replication, while the product of recF functioned as a recombinational DNA repair ATPase, serving multiple roles in DNA repair, homologous gene recombination, and DNA replication^[60]. And the mutS was responsible for mismatch repair^[61]. The upregulation of DnaN and DnaE encoding the chromosome replication initiation proteins at the mRNA level was observed, while the expression of RnhB and HolA was down regulated. Furthermore, the single-stranded DNA-binding protein SSB also played a crucial role in DNA replication, recombination, and damage repair processes^[62]. These results suggested that the biological processes could enable T. tengcongensis to survive at extreme temperatures by adapting to ambient temperature^[63].

Additionally, the rpmI, a gene involved in the ribosomal pathway, has been related to the thermotropism of T. tengcongensis^[64]. The ptsN3 encoding the mannitol/fructose-specific IIA structural domain of the phosphotransferase system (Ntr-type) exhibited a significant upregulation in transcript levels during thermophilic acclimatization, indicating its crucial involvement in the PTS^[65]. The primary energy source for the organism was typically derived from central carbon metabolism, which encompassed glycolysis, the TCA cycle, and the pentose phosphate pathway, providing essential precursor materials for other metabolic pathways^[66].

3.7 Induction of novel DEGs and DEGs with unknown function

In this study, a total of 16 specific DEGs were identified at 50, 60, 75, and 80 ℃. Among these, the ferritin gene napF3, which was involved in membrane lipid synthesis, also played an important role in different signaling pathways^{[3, 21]}. The rnhA was a ribonuclease HI gene associated with mouth-dependent chromosome replication^[67]. Furthermore, the ccmA2, a gene encoding a multidrug ABC transporter ATPase, was crucial for maintaining the normal morphology of bacteria and was closely related to the operation of bacteria^[68]. In addition, sRNA49, sRNA103, and sRNA104 were small RNAs that had the potential to modulate the expression of proteins related to thermophiles proteins^[69]. However, the names and functions of the remaining genes are unknown, and their regulatory mechanisms of thermal adaptation in thermophiles also remain unclear. These results suggested that these DEGs might interact with thiD at different temperature, thereby influencing the activity of ΔthiD and assuming other physiological functions in the process of thermal adaptation.

In conclusion, the thiD disrupted mutant did not grow at 80 ℃, and its growth rate at 50, 60, and 75 ℃ was slower than that of the WT strain, indicating that ThiD played a role in the normal growth, metabolism, and survival of T. tengcongensis. The transcriptome analyses indicated that the thermal adaptation of T. tengcongensis involved the coordinated regulation of various signaling pathways. It was observed that only a limited set of DEGs within each pathway were modified and involved in the regulation of thermal adaptation. However, additional empirical evidence is necessary to comprehensively elucidate T. tengcongensis to thrive in high-temperature environments. This study would provide new insights into the mechanisms of thermal adaptation in T. tengcongensis.

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2024年第64卷第9期

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doi: 10.13343/j.cnki.wsxb.20240153

接收时间：2024-03-10
首发时间：2026-03-20
出版时间：2024-05-30

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收稿日期：2024-03-10
录用日期：2024-05-28

基金

the Science and Technology Innovation Fund of Gansu Agricultural University (Young Mentor Support Fund)(GAU-QDFC-2023-04)

the National Natural Science Foundation of China(31500067)

作者信息

1 甘肃农业大学动物医学院, 甘肃兰州 730070

2 北京市畜牧总站, 北京 100101

3 天水师范学院, 甘肃天水 741000

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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