微生物学报

引物 Primers	核酸序列 Nucleotide sequences (5′→3′)	qPCR产物 Products of qPCR
1-F	CGGTACTGGCAAGATCGGTCTG	WP_265061909.1
1-R	TGATGTCGGATTGGCGGAACAG
2-F	GCAACCTGGTGGCAGTCATCT	WP_201954220.1
2-R	CTGGCTGGTCTCGTGCTTCA
3-F	TGCTCTGGTTGCCGCTGTTC	WP_005335875.1
3-R	ACCTGACCTATGGTGCCGATGG
4-F	ATGGCGGTGGATCTGGAGGT	WP_265062582.1
4-R	GCGGTCTTGAGGCTGATGGT
5-F	GCAGCAATGACTTCGTGACCAA	WP_265062605.1
5-R	CGGCAATCTCACCAGACTCTTC
6-F	AAGACCGTCATTCGTGCCAACT	WP_265061092.1
6-R	GCCAGCTTCTCTACCAGCAGTT
7-F	GTGTCTGGCAAGAGAAGTGGCT	WP_167566591.1
7-R	GCGTCGTGAGTGGCGTGATA
8-F	GCGGGACATCCTCTATGCCATA	WP_040066712.1
8-R	ATGGAACAGGCTGCTGTCACA
9-F	CCACCTGCGTTACACCTTCCA	WP_265061310.1
9-R	CAGTGCTTGCCAGCCATCGT
10-F	AGCCACACCAGCGTGATCCT	WP_265061484.1
10-R	CGCCTTGGTTCCAGCATGATGA
11-F	GCAAGCCTGTTCGTGGTGGTAT	WP_265061494.1
11-R	CAAGCGTGCCATCAGCGTGA
12-F	CCCACTGGCGAACAGGTTTG	WP_033136990.1
12-R	GAGGTCGGCAGGCTCAATCT
13-F	ACAACTGCTGGTGCTGGATGAA	WP_204481558.1
13-R	AGGCAAGGTGGCGGAGAAGA
14-F	ACGGTGACATTCTGGTGTGGAA	WP_201994931.1
14-R	ATCCTCGTCGCCTGTGATAGC
15-F	TGAAGATGGTGCTGCTGGACAA	WP_203119821.1
15-R	CGGAAAGGATGGTCTCGCTGTA

引物 Primers	核酸序列 Nucleotide sequences (5′→3′)	qPCR产物 Products of qPCR
1-F	CGGTACTGGCAAGATCGGTCTG	WP_265061909.1
1-R	TGATGTCGGATTGGCGGAACAG
2-F	GCAACCTGGTGGCAGTCATCT	WP_201954220.1
2-R	CTGGCTGGTCTCGTGCTTCA
3-F	TGCTCTGGTTGCCGCTGTTC	WP_005335875.1
3-R	ACCTGACCTATGGTGCCGATGG
4-F	ATGGCGGTGGATCTGGAGGT	WP_265062582.1
4-R	GCGGTCTTGAGGCTGATGGT
5-F	GCAGCAATGACTTCGTGACCAA	WP_265062605.1
5-R	CGGCAATCTCACCAGACTCTTC
6-F	AAGACCGTCATTCGTGCCAACT	WP_265061092.1
6-R	GCCAGCTTCTCTACCAGCAGTT
7-F	GTGTCTGGCAAGAGAAGTGGCT	WP_167566591.1
7-R	GCGTCGTGAGTGGCGTGATA
8-F	GCGGGACATCCTCTATGCCATA	WP_040066712.1
8-R	ATGGAACAGGCTGCTGTCACA
9-F	CCACCTGCGTTACACCTTCCA	WP_265061310.1
9-R	CAGTGCTTGCCAGCCATCGT
10-F	AGCCACACCAGCGTGATCCT	WP_265061484.1
10-R	CGCCTTGGTTCCAGCATGATGA
11-F	GCAAGCCTGTTCGTGGTGGTAT	WP_265061494.1
11-R	CAAGCGTGCCATCAGCGTGA
12-F	CCCACTGGCGAACAGGTTTG	WP_033136990.1
12-R	GAGGTCGGCAGGCTCAATCT
13-F	ACAACTGCTGGTGCTGGATGAA	WP_204481558.1
13-R	AGGCAAGGTGGCGGAGAAGA
14-F	ACGGTGACATTCTGGTGTGGAA	WP_201994931.1
14-R	ATCCTCGTCGCCTGTGATAGC
15-F	TGAAGATGGTGCTGCTGGACAA	WP_203119821.1
15-R	CGGAAAGGATGGTCTCGCTGTA

靶标 Target	结合区域 Interaction regions	结合能 Energy	P值 P-value	功能 Function
^a−f: Genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 match none of genes in A. veronii C4; −^*a−f: The biological functions of genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 are not annotated.
WP_040066712.1	253−337	−21.54	0.000 097 8	Cation symporter-2 family protein
WP_167566591.1	255−282	−20.72	0.000 227 0	Hypothetical protein
WP_204481558.1	271−337	−20.56	0.000 265 4	DEAD/DEAH box helicase
FOB40_RS11515^a	193−336	−20.09	0.000 414 3	−^*a
WP_005339652.1	299−337	−20.04	0.000 433 9	PfkB family carbohydrate kinase
WP_265062582.1	253−296	−19.41	0.000 763 3	Alpha-2-macroglobulin
WP_124929292.1	216−317	−19.29	0.000 847 0	Glutathione peroxidase
WP_265062250.1	266−294	−19.18	0.000 930 8	Methylated-DNA-[protein]-cysteine S-methyltransferase
WP_265061484.1	191−221	−18.95	0.001 130 7	ATP-dependent helicase HrpB
WP_265061867.1	313−337	−18.92	0.001 159 4	Glycosyltransferase family 9 protein
FOB40_RS04270^b	303−336	−18.87	0.001 208 8	−^*b
WP_201994931.1	271−322	−18.74	0.001 346 1	Aspartate-ammonia ligase
FOB40_RS17420^c	251−266	−18.62	0.001 485 0	−^*c
FOB40_RS07355^d	261−281	−18.53	0.001 597 6	−^*d
WP_191966279.1	136−162	−18.50	0.001 636 8	Rhombosortase
FOB40_RS05245^e	288−341	−18.16	0.002 145 4	−^*e
WP_108612743.1	271−325	−18.05	0.002 338 0	MarC family NAAT transporter
FOB40_RS01440^f	315−338	−17.57	0.003 373 3	−^*f
WP_265061975.1	198−219	−17.56	0.003 398 7	DgaE family pyridoxal phosphate-dependent ammonia lyase
WP_265062605.1	314−337	−17.48	0.003 607 9	Inositol-1-monophosphatase
WP_005348017.1	215−259	−17.44	0.003 716 8	GMP reductase
WP_265062619.1	37−63	−17.39	0.003 857 1	Alkene reductase
WP_005336914.1	290−338	−17.37	0.003 914 5	Anaerobic C4-dicarboxylate transporter DcuC
WP_265060904.1	213−323	−17.24	0.004 306 8	NADH-quinone oxidoreductase subunit H
WP_033136990.1	208−225	−17.07	0.004 872 9	Phage tail assembly protein

靶标 Target	结合区域 Interaction regions	结合能 Energy	P值 P-value	功能 Function
^a−f: Genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 match none of genes in A. veronii C4; −^*a−f: The biological functions of genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 are not annotated.
WP_040066712.1	253−337	−21.54	0.000 097 8	Cation symporter-2 family protein
WP_167566591.1	255−282	−20.72	0.000 227 0	Hypothetical protein
WP_204481558.1	271−337	−20.56	0.000 265 4	DEAD/DEAH box helicase
FOB40_RS11515^a	193−336	−20.09	0.000 414 3	−^*a
WP_005339652.1	299−337	−20.04	0.000 433 9	PfkB family carbohydrate kinase
WP_265062582.1	253−296	−19.41	0.000 763 3	Alpha-2-macroglobulin
WP_124929292.1	216−317	−19.29	0.000 847 0	Glutathione peroxidase
WP_265062250.1	266−294	−19.18	0.000 930 8	Methylated-DNA-[protein]-cysteine S-methyltransferase
WP_265061484.1	191−221	−18.95	0.001 130 7	ATP-dependent helicase HrpB
WP_265061867.1	313−337	−18.92	0.001 159 4	Glycosyltransferase family 9 protein
FOB40_RS04270^b	303−336	−18.87	0.001 208 8	−^*b
WP_201994931.1	271−322	−18.74	0.001 346 1	Aspartate-ammonia ligase
FOB40_RS17420^c	251−266	−18.62	0.001 485 0	−^*c
FOB40_RS07355^d	261−281	−18.53	0.001 597 6	−^*d
WP_191966279.1	136−162	−18.50	0.001 636 8	Rhombosortase
FOB40_RS05245^e	288−341	−18.16	0.002 145 4	−^*e
WP_108612743.1	271−325	−18.05	0.002 338 0	MarC family NAAT transporter
FOB40_RS01440^f	315−338	−17.57	0.003 373 3	−^*f
WP_265061975.1	198−219	−17.56	0.003 398 7	DgaE family pyridoxal phosphate-dependent ammonia lyase
WP_265062605.1	314−337	−17.48	0.003 607 9	Inositol-1-monophosphatase
WP_005348017.1	215−259	−17.44	0.003 716 8	GMP reductase
WP_265062619.1	37−63	−17.39	0.003 857 1	Alkene reductase
WP_005336914.1	290−338	−17.37	0.003 914 5	Anaerobic C4-dicarboxylate transporter DcuC
WP_265060904.1	213−323	−17.24	0.004 306 8	NADH-quinone oxidoreductase subunit H
WP_033136990.1	208−225	−17.07	0.004 872 9	Phage tail assembly protein

靶标基因 Target gene	注释 Annotation
WP_265061909.1	2-hydroxyacid dehydrogenase
WP_201954220.1	Malate dehydrogenase
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase
WP_265062582.1	Penicillin-binding protein 1C
WP_265062605.1	Inositol-1-monophosphatase
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase
WP_167566591.1	Derived by automated computational analysis using gene prediction method: protein homology.
WP_040066712.1	Nucleobase: cation symporter-2 family protein
WP_265061310.1	Bifunctional metallophosphatase/5′-nucleotidase
WP_265061484.1	ATP-dependent helicase HrpB
WP_265061494.1	d-hexose-6-phosphate mutarotase
WP_033136990.1	Phage tail assembly protein
WP_204481558.1	DEAD/DEAH box helicase
WP_201994931.1	Aspartate-ammonia ligase
WP_203119821.1	Acetolactate synthase 2 small subunit

靶标基因 Target gene	注释 Annotation
WP_265061909.1	2-hydroxyacid dehydrogenase
WP_201954220.1	Malate dehydrogenase
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase
WP_265062582.1	Penicillin-binding protein 1C
WP_265062605.1	Inositol-1-monophosphatase
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase
WP_167566591.1	Derived by automated computational analysis using gene prediction method: protein homology.
WP_040066712.1	Nucleobase: cation symporter-2 family protein
WP_265061310.1	Bifunctional metallophosphatase/5′-nucleotidase
WP_265061484.1	ATP-dependent helicase HrpB
WP_265061494.1	d-hexose-6-phosphate mutarotase
WP_033136990.1	Phage tail assembly protein
WP_204481558.1	DEAD/DEAH box helicase
WP_201994931.1	Aspartate-ammonia ligase
WP_203119821.1	Acetolactate synthase 2 small subunit

基因编号 Gene ID	注释 Annotation	ΔssrA vs. WT (fold change)	ΔsmpB vs. WT (fold change)
WT indicates wild type, ΔssrA indicates tmRNA knockout strain, ΔsmpB indicates smpB knockout strain, −^*a−g indicates no significant difference.
WP_201994931.1	Aspartate-ammonia ligase	2.0	−^*g
WP_201954220.1	Malate dehydrogenase	−^*a	2.4
WP_265062582.1	Penicillin-binding protein 1C	−^*b	2.3
WP_265061484.1	ATP-dependent helicase HrpB	−^*c	3.5
WP_265061494.1	d-hexose-6-phosphate mutarotase	−^*d	4.7
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase	−^*e	3.3
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase	−^*f	0.3

基因编号 Gene ID	注释 Annotation	ΔssrA vs. WT (fold change)	ΔsmpB vs. WT (fold change)
WT indicates wild type, ΔssrA indicates tmRNA knockout strain, ΔsmpB indicates smpB knockout strain, −^*a−g indicates no significant difference.
WP_201994931.1	Aspartate-ammonia ligase	2.0	−^*g
WP_201954220.1	Malate dehydrogenase	−^*a	2.4
WP_265062582.1	Penicillin-binding protein 1C	−^*b	2.3
WP_265061484.1	ATP-dependent helicase HrpB	−^*c	3.5
WP_265061494.1	d-hexose-6-phosphate mutarotase	−^*d	4.7
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase	−^*e	3.3
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase	−^*f	0.3

维氏气单胞菌tmRNA以sRNA形式特异调控的下游靶标筛选和鉴定

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童园 , 罗宸 , 柏泰鹏 , 唐燕琼 , 刘柱 , 马香 ^*

微生物学报 | 研究报告 2024,64(9): 3282-3294

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微生物学报 | 研究报告 2024, 64(9): 3282-3294

维氏气单胞菌tmRNA以sRNA形式特异调控的下游靶标筛选和鉴定

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童园, 罗宸, 柏泰鹏, 唐燕琼, 刘柱, 马香^*

作者信息

海南大学生命健康学院, 海南海口 570228

Screening and identification of downstream targets specifically regulated by Aeromonas veronii tmRNA in the form of sRNA

Yuan TONG, Chen LUO, Taipeng BAI, Yanqiong TANG, Zhu LIU, Xiang MA^*

Affiliations

School of Life and Health Sciences, Hainan University, Haikou 570228, Hainan, China

出版时间: 2024-06-04 doi: 10.13343/j.cnki.wsxb.20240107

文章导航

摘要

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转移-信使RNA (transfer-message RNA, tmRNA)是细菌中普遍存在的稳定非编码sRNA，同时具备tRNA和mRNA双重特性，主要介导反式翻译核糖体拯救机制，对病原菌致病性和胁迫响应具有重要影响。【目的】从tmRNA功能研究入手，揭示对水产养殖和人类公共安全造成严重危害的维氏气单胞菌(Aeromonas veronii)的致病分子机制。【方法】采用生物学信息学工具IntaRNA 2.0对维氏气单胞菌tmRNA可能结合的下游靶标进行预测，通过基因本体论(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)富集分析，鉴定预测靶标参与的生物学过程及信号通路，并使用qPCR技术验证候选靶标基因在维氏气单胞菌野生型、tmRNA敲除株和smpB敲除株中的表达情况，以初步鉴定维氏气单胞菌tmRNA以sRNA形式调控的潜在下游靶标。【结果】tmRNA可以sRNA形式与下游100个潜在特异性靶标结合，这些靶标基因主要结合于tmRNA的3′端tRNA样结构域(tRNA-like domain, TLD)、H2域及PK3、PK4域，参与细菌基本代谢过程。qPCR结果表明，在特异性结合的靶标基因中，基因WP_201994931.1以SmpB非依赖的方式受到tmRNA调节；WP_201954220.1、WP_005335875.1、WP_265062582.1、WP_265061484.1和WP_265061494.1等基因的表达则受到SmpB蛋白调控。【结论】本研究初步鉴定基因WP_201994931.1有可能是tmRNA作为sRNA调控的下游靶标，对拓展tmRNA功能研究领域有一定指导作用，有助于后续进一步探究维氏气单胞菌的致病和环境适应等重要生命活动的分子机制。

关键词

维氏气单胞菌 / tmRNA / sRNA / 特异调控下游靶标

Abstract

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Transfer-message RNA (tmRNA) is a ubiquitous and stable non-coding small RNA in bacteria, with characteristic regions similar with both tRNA and mRNA. tmRNA is known to mediate the ribosome rescue mechanism called trans-translation and has effects on the pathogenicity and stress responses of pathogenic bacteria. [Objective] To study the function of tmRNA in Aeromonas veronii causing threats to aquaculture and human public safety, so as to reveal the molecular mechanism for the pathogenicity of A. veronii. [Methods] IntaRNA 2.0 was used to predict the downstream targets binding to tmRNA. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted to predict the biological processes and signaling pathways involving the predicted targets. Real-time qPCR was employed to compare the expression levels of the candidate target genes among the wild type, tmRNA knockout strain, and smpB knockout strain of A. veronii, thus identifying the potential targets regulated by tmRNA in the form of sRNA. [Results] One hundred potential specific downstream targets might bind to tmRNA at the 3′-end tRNA-like domain (tRNA-like domain, TLD), H2 domain, and PK3 and PK4 regions, thus participating in the general metabolic pathways of the pathogen. The results of qPCR indicated that the expression of WP_201994931.1 was regulated by tmRNA in an SmpB-independent manner, while the expression of WP_201954220.1, WP_005335875.1, WP_265062582.1, WP_265061484.1, and WP_265061494.1 was regulated by SmpB. [Conclusion] We preliminarily identified that WP_201994931.1 might constitute a downstream target regulated by tmRNA as an sRNA. This study provides basic information for expanding the functions of tmRNA in the form of sRNA and facilitates the further studies regarding the molecular mechanisms of the pathogenicity and environmental adaptation of A. veronii.

Key words

Aeromonas veronii / tmRNA / sRNA / specifically regulated targets

引用本文

童园, 罗宸, 柏泰鹏, 唐燕琼, 刘柱, 马香. 维氏气单胞菌tmRNA以sRNA形式特异调控的下游靶标筛选和鉴定. 微生物学报, 2024 , 64 (9) : 3282 -3294 . DOI: 10.13343/j.cnki.wsxb.20240107

Yuan TONG, Chen LUO, Taipeng BAI, Yanqiong TANG, Zhu LIU, Xiang MA. Screening and identification of downstream targets specifically regulated by Aeromonas veronii tmRNA in the form of sRNA[J]. Acta Microbiologica Sinica, 2024 , 64 (9) : 3282 -3294 . DOI: 10.13343/j.cnki.wsxb.20240107

正文

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非编码小RNA (no-coding small RNA, sRNA)在细菌中普遍存在，长度在50−500 nt之间，具有基因表达调控活性^[1-2]，影响细菌响应外界胁迫和发挥致病性^[3]。当前对于sRNA及其介导的基因表达调控的研究是阐释细菌致病性的重要切入点^[4-5]。转移-信使RNA (transfer-message RNA, tmRNA)是细菌中普遍存在的稳定非编码小RNA，由ssrA基因编码，长度约260−430 bp^[6]。成熟tmRNA的二级结构由tRNA样结构域、mRNA样结构域(MLD)、4个伪结结构(PK1−PK4)以及2个螺旋结构域(H2和H5)组成^[7]，其主要作为“反式翻译(trans-translation)”系统的核心成分，与核心蛋白small protein B (SmpB)协同释放阻滞的核糖体，保障蛋白质合成^[8-9]。tmRNA或SmpB的缺失都可导致反式翻译缺陷，引发细菌表型变化^[10]，影响细菌的生长增殖、胁迫耐受、抗生素耐受和致病性等^[11-13]。近年的研究揭示，tmRNA极有可能以sRNA形式发挥功能，通过靶向特定靶标调控细菌的环境适应力，如铁离子代谢、氧化应激管理^[14]等，还包括对抗生素^[15]和有机溶剂的耐受性^[16]。然而，关于tmRNA作为反义sRNA发挥功能的研究几乎空白。目前已在大肠杆菌(Escherichia coli)中初步证明，tmRNA通过调控lac操纵子转录活性而调节细菌的乳糖代谢^[17]；同时，在金黄色葡萄球菌(Staphylococcus aureus)中，tmRNA以不依赖于反式翻译系统的方式，靶向色素合成关键酶crtMN mRNA，通过调控色素代谢而介导氧化耐受^[18]。以上研究暗示tmRNA以sRNA分子形式发挥功能调控细菌代谢的巨大潜力，一定程度上解释了在某些细菌中tmRNA和SmpB缺失导致表型不一致的原因^[10]。然而，tmRNA如何在参与反式翻译与作为sRNA调控分子两种作用机制之间转换，以及其作为sRNA时调控的靶标基因还有待进一步研究。

细菌性疾病是危害鱼类养殖业发展的重要因素之一，其中由维氏气单胞菌(Aeromonas veronii)引发的病害问题尤为显著^[19]。维氏气单胞菌属革兰氏阴性杆状细菌，广泛存在于淡水、污水等环境中^[20]。该致病菌的宿主范围十分广泛，包括鱼类、节肢动物和爬行类动物等，能够引发多种感染病症^{[19, 21]}，对水产经济发展和人类健康构成严重威胁^[22-23]。为了深入解析维氏气单胞菌C4的环境适应和致病机制，本研究首先采用sRNA靶标预测软件，预测维氏气单胞菌tmRNA可能以sRNA形式结合的潜在下游靶标；接着，对预测出的靶标进行富集分析，成功筛选出15个候选靶标基因；随后，通过qPCR验证了维氏气单胞菌C4 WT (野生型)、ΔssrA (ssrA敲除株)及ΔsmpB (smpB敲除株)中这些候选靶标的表达情况，从而鉴定了维氏气单胞菌C4中由tmRNA以sRNA形式调控的潜在下游靶标。

本研究结果不仅有助于后续进一步探索tmRNA以sRNA形式发挥的功能，拓展tmRNA功能研究领域，还有望深化sRNA调控机制的研究，对指导后续深入研究具有重要的理论意义。

1 材料与方法

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1.1 主要试剂和仪器

核酸染料Super Red、电泳上样缓冲液6×DNA Loading Buffer，北京兰杰柯科技有限公司；GN5K DNA Marker，武汉赛维尔生物科技有限公司；2×Rapid Taq Master Mix、5×HiScript III qRT SuperMix、2×ChamQ SYBR Color Master Mix，南京诺唯赞生物科技股份有限公司；Sodium Lauryl Sulfate (SDS)、卡那霉素(kanamycin)、氨苄青霉素(ampicillin)，北京索莱宝科技有限公司；细菌总RNA快速抽提试剂盒，生工生物工程(上海)股份有限公司。

全温培养摇床，宁波江南仪器厂；生化培养箱，上海博迅医疗生物仪器股份有限公司；超净工作台，上海精密科学仪器有限公司；qPCR仪，Roche Diagnostics公司。

1.2 菌株

本研究所用菌株A. veronii C4 (野生型，氨苄青霉素抗性)、ΔssrA (tmRNA基因敲除株，氨苄青霉素与卡那霉素抗性)及ΔsmpB (smpB基因敲除株，氨苄青霉素抗性)均为本实验室保存菌株。

1.3 RNA提取及逆转录

使用细菌总RNA快速试剂盒[生工生物工程(上海)股份有限公司]提取菌株RNA：取1 mL对数生长期的细菌置于1.5 mL离心管中，4 ℃、8 000 r/min离心1 min，弃培养基，加入100 µL溶菌酶(浓度为400 µg/mL)，振荡混匀；加入900 µL Buffer Rlysis-B振荡混匀，室温放置3 min后，向裂解样品中加入200 µL氯仿，充分混匀，4 ℃、12 000 r/min离心5 min，取上清；向上清液中加入250 µL无水乙醇，混匀，室温放置3 min，4 ℃、12 000 r/min离心5 min，弃上清；取700 µL的75%乙醇(用DEPC-treated ddH₂O与无水乙醇配制)洗涤离心管底部沉淀，4 ℃、12 000 r/min离心3 min，弃上清；倒置离心管10 min，尽可能使离心管中残留的乙醇彻底挥发；最后加入30−50 µL DEPC-treated ddH₂O溶解离心管底部沉淀的RNA；检测RNA浓度后(进行凝胶电泳验证提取的RNA质量)，立即使用或放置−80 ℃冰箱中长期保存。

利用RT-PCR扩增提取的RNA基因序列。RT-PCR反应体系(10 μL)：5×HiScript III qRT SuperMix 2 µL，RNA模板500 ng，DEPC-treated ddH₂O补足至10 µL。RT-PCR反应条件：50 ℃逆转录15 min，85 ℃稳定30 s。逆转录获得的cDNA置于4 ℃短期保存，长期保存则置于–80 ℃。

1.4 实时荧光定量PCR (qPCR)

以逆转录获得的cDNA为模板进行qPCR。引物序列见表1，以梯度稀释的cDNA为模板，验证所使用引物的扩增效率。qPCR反应体系：2×ChamQ SYBR Color Master Mix 5 μL，上、下游引物(10 µmol/L)各0.4 µL，cDNA 1 μL，ddH₂O 3.2 μL (体系中所用cDNA模板均取用100 μL DEPC-treated ddH₂O稀释)。qPCR反应条件：95 ℃预变性30 s；95 ℃变性10 s，60 ℃退火 & 延伸(采集荧光信号) 30 s，40个循环；使用qPCR仪采集引物溶解曲线。采用2^−ΔΔC_t方法计算基因相对表达量^[24]。

1.5 tmRNA靶标基因预测

使用sRNA靶标预测软件IntaRNA 2.0进行维氏气单胞菌tmRNA靶标预测，sRNA靶标基因预测网址(IntaRNA 2.0)：http://rna.informatik.uni-freiburg.de/IntaRNA；基因序列获取网址(NCBI)：https://www.ncbi.nlm.nih.gov/gene/60844631。

1.6 GO和KEGG富集分析

使用OmicShare基迪奥生物信息云平台进行预测靶标的基因本体论(gene ontology, GO)富集分析与京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)富集分析。GO富集分析网址：https://www.omicshare.com/tools/Home/Soft/gogseasenior；KEGG富集分析网址：https://www.omicshare.com/tools/Home/Soft/pathwaygseasenior。

1.7 统计学分析

统计分析用SPSSAU软件进行，使用独立样本t检验分析实验结果的差异显著性，并用WPS绘制柱状图。以P < 0.05为差异具有统计学意义，*表示0.01 < P < 0.05，**表示P < 0.01，***表示P < 0.001，ns表示差异无统计学意义。

2 结果与分析

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2.1 tmRNA特异调控下游靶标预测

本研究获得了100个潜在靶标。表2列出按照P-value值排序的前25个潜在靶标，并参考维氏气单胞菌(A. veronii) C4的基因组序列(GenBank登录号为CP110364.1)标注预测的靶标基因编号。通过分析上述潜在靶标在tmRNA结构上的预测结合区域，发现潜在靶标虽有可能结合于tmRNA的各区段，但互作位点主要集中分布在tmRNA核苷酸序列的200−360 bp之间(图1)；进一步观察其二级结构发现，主要的结合位点分布在tmRNA的3′端tRNA样域、H2域及PK3、PK4区域(图2)。预测靶标的结合能及P-value数值越低，表明结合后的稳定性相对越高，结合能力也越强。结合表2中的数据，表明tmRNA与其预测靶标之间存在较强的结合力。

2.2 tmRNA特异调控下游靶标GO和KEGG富集分析

将100个潜在的维氏气单胞菌tmRNA特异性调控下游靶标进行GO和KEGG富集分析后，发现预测的靶标基因主要参与调控维氏气单胞菌的基本代谢、催化酶的代谢以及结合功能(图3)。进一步对显著富集的靶标基因进行分析发现，其主要集中于维氏气单胞菌的代谢通路中(图4)。为了进行后续验证分析，从显著富集的各通路中选择了具有代表性的靶标基因，并结合参考预测的P-value值，最终筛选出15个候选靶标基因(表3)。

2.3 实时荧光定量PCR (qPCR)检测候选靶标基因表达特异性

参考维氏气单胞菌(A. veronii) C4的基因组序列(GenBank登录号为CP110364.1)，为表5中列出的15个候选靶标基因设计了特异性qPCR引物。以细菌基因组为模板，通过PCR验证上述引物的有效性[附图1 (S1)，数据已提交国家微生物科学数据中心，编号为NMDCX0000291]。为后续进行qPCR验证，提取了质量优良的稳定RNA [附图2 (S2)，数据已提交国家微生物科学数据中心，编号为NMDCX0000292]；以逆转录获得的cDNA梯度稀释样品为模板，验证了15对qPCR引物(表1)的扩增效率。结果表明，这15对引物的扩增效率较高，均达到了90%−110%的范围[附图3 (S3)，数据已提交国家微生物科学数据中心，编号为NMDCX0000293]。

鉴于tmRNA以不依赖于SmpB的方式特异调控下游靶标^[18]，通过qPCR实验比较了候选基因在维氏气单胞菌WT、ΔssrA及ΔsmpB菌株中的表达差异，以初步鉴定tmRNA特异调控的下游靶标。如图5所示，以16S rRNA编码基因为内参，与WT相比，ΔssrA中基因WP_201994931.1的表达水平显著上调2.0倍，而在ΔsmpB中，该基因表达量与WT相对一致，表明基因WP_201994931.1极有可能是tmRNA以sRNA形式、不依赖于SmpB蛋白所调控的下游靶标。

qPCR结果表明，敲除smpB基因后，基因WP_201954220.1、WP_265062582.1、WP_265061484.1与WP_265061494.1的表达水平分别显著上调2.4、2.3、3.5和4.7倍，基因WP_005335875.1的表达水平极显著上调3.3倍，而基因WP_265061092.1的表达水平极显著下降，为野生型的0.3倍，相较于ΔssrA，上述基因与WT的相对表达量差异并不显著。上述结果表明，大部分与tmRNA特异结合的靶标基因的表达都具有SmpB依赖性(图5，表4)。

3 讨论与结论

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研究提示，tmRNA以sRNA的形式独立于反式翻译系统的另一核心元件SmpB，通过碱基互补配对原则特异性结合其靶基因，形成分子调控网络，参与细胞的代谢过程。Liu等研究表明，tmRNA作为反义RNA，通过调节crt-MN的表达来抑制金黄色葡萄球菌类胡萝卜素的合成^[17]。本课题组Wang等的研究发现，在维氏气单胞菌ΔssrA菌株中存在不需要SmpB参与的tmRNA介导的生理过程：在ΔssrA菌株中，次黄嘌呤合成和鸟苷代谢相关酶的表达显著提高，最终导致代谢产物次黄嘌呤的增加，从而影响细菌对甲氧苄啶的耐药性；同时，ssrA的敲除也使外排泵AcrA/AcrB的表达下调，这可能导致菌株对甲氧苄啶更为敏感，但在ΔsmpB菌株中并未观察到相同的现象^[25]。这些报道与本研究的GO和KEGG富集分析结果一致，说明tmRNA的靶基因在细菌代谢过程中发挥重要作用。

根据本研究的qPCR结果分析，相较于WT，基因WP_201994931.1 (asnA)在ΔssrA中表达量显著提高，而在smpB缺失的情况下无显著变化，表明WP_201994931.1以非依赖SmpB的方式受到tmRNA调控^[26]。已知SmpB蛋白是tmRNA介导的反式翻译过程所必需的，上述结果则暗示tmRNA可能以独立于反式翻译机制的sRNA形式负调控WP_201994931.1的表达。WP_201994931.1编码天冬酰胺合成酶A (asparagine synthetase A，AS-A)，该酶能够调节天冬氨酸的生物合成，并通过激活ATP依赖性氨基转移酶催化天冬氨酸及谷氨酰胺的生物合成^[27-28]。AsnA参与调控氨基酸生物合成，丙氨酸、天冬氨酸和谷氨酸代谢、氰基氨基酸代谢等代谢通路，而tmRNA可能以sRNA形式参与调控上述生物过程。

本研究还发现部分预测与tmRNA结合的靶标基因表达量在ΔsmpB菌株中的表达量显著变化，但在tmRNA缺失的情况下这些变化不受影响。基因WP_265061743.1、WP_005335875.1、WP_265062582.1、WP_265061484.1与WP_265061494.1在ΔsmpB菌株中表达水平显著上调，而WP_265061092.1基因在ΔsmpB菌株中的表达水平极显著下降。然而，在ΔssrA菌株中，这些基因的表达量并未观察到显著差异。这一结果可能是由于维氏气单胞菌中smpB基因在稳定期的表达量较指数期升高，而tmRNA表达量在指数期和稳定期均保持稳定，这暗示反式翻译系统主要通过改变SmpB蛋白的合成量而调控自身功能活性。研究表明，SmpB蛋白可能通过直接或间接方式调控沙门氏菌属(Salmonella)中至少4%的下游基因表达，从而影响病原菌的毒力^[29-30]。因此，维氏气单胞菌中的SmpB蛋白也可能以独立于反式翻译的机制调控上述基因表达。当然，由于本研究仅通过qPCR检测了靶标基因的mRNA水平的变化，其编码蛋白产物的变化情况待通过制备抗体或添加标签进行免疫印迹实验进一步验证。

在本研究中，基因WP_201954220.1编码的苹果酸脱氢酶(malate dehydrogenase, MDH)是三羧酸循环的关键氧化还原酶，参与调控C4循环、糖异生、脂肪酸氧化、氮同化和氨基酸生物合成等生理生化反应^[31]。基因WP_005335875.1编码的1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferases, AGPATs)是甘油磷脂(glycerophospholipid, GPL)和三酰基甘油(triacylglycerol, TAG)生物合成途径的中间酶，参与甘油磷脂及甘油脂代谢，与人类的糖尿病等疾病相关^[32]。基因WP_265061494.1编码的d-己糖-6-磷酸变旋酶能催化3种6-磷酸己糖(glucose 6-phosphate, Glc6P; galactosamine 6-phosphate, Gal6P; mannose 6-phosphate, Man6P)的α和β异构体之间的相互转换，参与糖酵解、糖异生作用^[33]。基因WP_265061092.1编码一种芳香族氨基酸转氨酶(aromatic amino acid transaminase, ARAT)，通过催化苯丙氨酸、酪氨酸、天冬氨酸和亮氨酸的氨基转移，参与氨基酸合成与分解、碳氮吸收、次级代谢等重要代谢过程^[34]。上述基因的表达量均受到smpB特异性调控，表明SmpB可能通过反式翻译或独立于反式翻译外的其他方式调控相关的代谢通路，进而影响细菌的正常生命活动。

本研究的结果对于指导后续深入研究tmRNA以sRNA形式调控的特异性下游靶标，或SmpB单独或依赖反式翻译调控的下游靶标及其功能和通路，进而进一步解析维氏气单胞菌的基本生命活动和致病机制等，均具有重要理论意义。

基金

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海南省自然科学基金(320RC502)

参考文献

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2024年第64卷第9期

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doi: 10.13343/j.cnki.wsxb.20240107

接收时间：2024-02-20
首发时间：2026-03-20
出版时间：2024-06-04

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收稿日期：2024-02-20
录用日期：2024-05-31

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Natural Science Foundation of Hainan Province(320RC502)

海南省自然科学基金(320RC502)

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海南大学生命健康学院, 海南海口 570228

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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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引物 Primers	核酸序列 Nucleotide sequences (5′→3′)	qPCR产物 Products of qPCR
1-F	CGGTACTGGCAAGATCGGTCTG	WP_265061909.1
1-R	TGATGTCGGATTGGCGGAACAG
2-F	GCAACCTGGTGGCAGTCATCT	WP_201954220.1
2-R	CTGGCTGGTCTCGTGCTTCA
3-F	TGCTCTGGTTGCCGCTGTTC	WP_005335875.1
3-R	ACCTGACCTATGGTGCCGATGG
4-F	ATGGCGGTGGATCTGGAGGT	WP_265062582.1
4-R	GCGGTCTTGAGGCTGATGGT
5-F	GCAGCAATGACTTCGTGACCAA	WP_265062605.1
5-R	CGGCAATCTCACCAGACTCTTC
6-F	AAGACCGTCATTCGTGCCAACT	WP_265061092.1
6-R	GCCAGCTTCTCTACCAGCAGTT
7-F	GTGTCTGGCAAGAGAAGTGGCT	WP_167566591.1
7-R	GCGTCGTGAGTGGCGTGATA
8-F	GCGGGACATCCTCTATGCCATA	WP_040066712.1
8-R	ATGGAACAGGCTGCTGTCACA
9-F	CCACCTGCGTTACACCTTCCA	WP_265061310.1
9-R	CAGTGCTTGCCAGCCATCGT
10-F	AGCCACACCAGCGTGATCCT	WP_265061484.1
10-R	CGCCTTGGTTCCAGCATGATGA
11-F	GCAAGCCTGTTCGTGGTGGTAT	WP_265061494.1
11-R	CAAGCGTGCCATCAGCGTGA
12-F	CCCACTGGCGAACAGGTTTG	WP_033136990.1
12-R	GAGGTCGGCAGGCTCAATCT
13-F	ACAACTGCTGGTGCTGGATGAA	WP_204481558.1
13-R	AGGCAAGGTGGCGGAGAAGA
14-F	ACGGTGACATTCTGGTGTGGAA	WP_201994931.1
14-R	ATCCTCGTCGCCTGTGATAGC
15-F	TGAAGATGGTGCTGCTGGACAA	WP_203119821.1
15-R	CGGAAAGGATGGTCTCGCTGTA

引物
Primers

核酸序列
Nucleotide sequences (5′→3′)

qPCR产物
Products of qPCR

1-F

CGGTACTGGCAAGATCGGTCTG

WP_265061909.1

1-R

TGATGTCGGATTGGCGGAACAG

2-F

GCAACCTGGTGGCAGTCATCT

WP_201954220.1

2-R

CTGGCTGGTCTCGTGCTTCA

3-F

TGCTCTGGTTGCCGCTGTTC

WP_005335875.1

3-R

ACCTGACCTATGGTGCCGATGG

4-F

ATGGCGGTGGATCTGGAGGT

WP_265062582.1

4-R

GCGGTCTTGAGGCTGATGGT

5-F

GCAGCAATGACTTCGTGACCAA

WP_265062605.1

5-R

CGGCAATCTCACCAGACTCTTC

6-F

AAGACCGTCATTCGTGCCAACT

WP_265061092.1

6-R

GCCAGCTTCTCTACCAGCAGTT

7-F

GTGTCTGGCAAGAGAAGTGGCT

WP_167566591.1

7-R

GCGTCGTGAGTGGCGTGATA

8-F

GCGGGACATCCTCTATGCCATA

WP_040066712.1

8-R

ATGGAACAGGCTGCTGTCACA

9-F

CCACCTGCGTTACACCTTCCA

WP_265061310.1

9-R

CAGTGCTTGCCAGCCATCGT

10-F

AGCCACACCAGCGTGATCCT

WP_265061484.1

10-R

CGCCTTGGTTCCAGCATGATGA

11-F

GCAAGCCTGTTCGTGGTGGTAT

WP_265061494.1

11-R

CAAGCGTGCCATCAGCGTGA

12-F

CCCACTGGCGAACAGGTTTG

WP_033136990.1

12-R

GAGGTCGGCAGGCTCAATCT

13-F

ACAACTGCTGGTGCTGGATGAA

WP_204481558.1

13-R

AGGCAAGGTGGCGGAGAAGA

14-F

ACGGTGACATTCTGGTGTGGAA

WP_201994931.1

14-R

ATCCTCGTCGCCTGTGATAGC

15-F

TGAAGATGGTGCTGCTGGACAA

WP_203119821.1

15-R

CGGAAAGGATGGTCTCGCTGTA

靶标 Target	结合区域 Interaction regions	结合能 Energy	P值 P-value	功能 Function
^a−f: Genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 match none of genes in A. veronii C4; −^*a−f: The biological functions of genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 are not annotated.
WP_040066712.1	253−337	−21.54	0.000 097 8	Cation symporter-2 family protein
WP_167566591.1	255−282	−20.72	0.000 227 0	Hypothetical protein
WP_204481558.1	271−337	−20.56	0.000 265 4	DEAD/DEAH box helicase
FOB40_RS11515^a	193−336	−20.09	0.000 414 3	−^*a
WP_005339652.1	299−337	−20.04	0.000 433 9	PfkB family carbohydrate kinase
WP_265062582.1	253−296	−19.41	0.000 763 3	Alpha-2-macroglobulin
WP_124929292.1	216−317	−19.29	0.000 847 0	Glutathione peroxidase
WP_265062250.1	266−294	−19.18	0.000 930 8	Methylated-DNA-[protein]-cysteine S-methyltransferase
WP_265061484.1	191−221	−18.95	0.001 130 7	ATP-dependent helicase HrpB
WP_265061867.1	313−337	−18.92	0.001 159 4	Glycosyltransferase family 9 protein
FOB40_RS04270^b	303−336	−18.87	0.001 208 8	−^*b
WP_201994931.1	271−322	−18.74	0.001 346 1	Aspartate-ammonia ligase
FOB40_RS17420^c	251−266	−18.62	0.001 485 0	−^*c
FOB40_RS07355^d	261−281	−18.53	0.001 597 6	−^*d
WP_191966279.1	136−162	−18.50	0.001 636 8	Rhombosortase
FOB40_RS05245^e	288−341	−18.16	0.002 145 4	−^*e
WP_108612743.1	271−325	−18.05	0.002 338 0	MarC family NAAT transporter
FOB40_RS01440^f	315−338	−17.57	0.003 373 3	−^*f
WP_265061975.1	198−219	−17.56	0.003 398 7	DgaE family pyridoxal phosphate-dependent ammonia lyase
WP_265062605.1	314−337	−17.48	0.003 607 9	Inositol-1-monophosphatase
WP_005348017.1	215−259	−17.44	0.003 716 8	GMP reductase
WP_265062619.1	37−63	−17.39	0.003 857 1	Alkene reductase
WP_005336914.1	290−338	−17.37	0.003 914 5	Anaerobic C4-dicarboxylate transporter DcuC
WP_265060904.1	213−323	−17.24	0.004 306 8	NADH-quinone oxidoreductase subunit H
WP_033136990.1	208−225	−17.07	0.004 872 9	Phage tail assembly protein

靶标
Target

结合区域
Interaction regions

结合能
Energy

P值
P-value

功能
Function

^a−f: Genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 match none of genes in A. veronii C4; −^*a−f: The biological functions of genes FOB40_RS11515, FOB40_RS04270, FOB40_RS17420, FOB40_RS07355, FOB40_RS05245, and FOB40_RS01440 are not annotated.

WP_040066712.1

253−337

−21.54

0.000 097 8

Cation symporter-2 family protein

WP_167566591.1

255−282

−20.72

0.000 227 0

Hypothetical protein

WP_204481558.1

271−337

−20.56

0.000 265 4

DEAD/DEAH box helicase

FOB40_RS11515^a

193−336

−20.09

0.000 414 3

−^*a

WP_005339652.1

299−337

−20.04

0.000 433 9

PfkB family carbohydrate kinase

WP_265062582.1

253−296

−19.41

0.000 763 3

Alpha-2-macroglobulin

WP_124929292.1

216−317

−19.29

0.000 847 0

Glutathione peroxidase

WP_265062250.1

266−294

−19.18

0.000 930 8

Methylated-DNA-[protein]-cysteine S-methyltransferase

WP_265061484.1

191−221

−18.95

0.001 130 7

ATP-dependent helicase HrpB

WP_265061867.1

313−337

−18.92

0.001 159 4

Glycosyltransferase family 9 protein

FOB40_RS04270^b

303−336

−18.87

0.001 208 8

−^*b

WP_201994931.1

271−322

−18.74

0.001 346 1

Aspartate-ammonia ligase

FOB40_RS17420^c

251−266

−18.62

0.001 485 0

−^*c

FOB40_RS07355^d

261−281

−18.53

0.001 597 6

−^*d

WP_191966279.1

136−162

−18.50

0.001 636 8

Rhombosortase

FOB40_RS05245^e

288−341

−18.16

0.002 145 4

−^*e

WP_108612743.1

271−325

−18.05

0.002 338 0

MarC family NAAT transporter

FOB40_RS01440^f

315−338

−17.57

0.003 373 3

−^*f

WP_265061975.1

198−219

−17.56

0.003 398 7

DgaE family pyridoxal phosphate-dependent ammonia lyase

WP_265062605.1

314−337

−17.48

0.003 607 9

Inositol-1-monophosphatase

WP_005348017.1

215−259

−17.44

0.003 716 8

GMP reductase

WP_265062619.1

37−63

−17.39

0.003 857 1

Alkene reductase

WP_005336914.1

290−338

−17.37

0.003 914 5

Anaerobic C4-dicarboxylate transporter DcuC

WP_265060904.1

213−323

−17.24

0.004 306 8

NADH-quinone oxidoreductase subunit H

WP_033136990.1

208−225

−17.07

0.004 872 9

Phage tail assembly protein

靶标基因 Target gene	注释 Annotation
WP_265061909.1	2-hydroxyacid dehydrogenase
WP_201954220.1	Malate dehydrogenase
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase
WP_265062582.1	Penicillin-binding protein 1C
WP_265062605.1	Inositol-1-monophosphatase
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase
WP_167566591.1	Derived by automated computational analysis using gene prediction method: protein homology.
WP_040066712.1	Nucleobase: cation symporter-2 family protein
WP_265061310.1	Bifunctional metallophosphatase/5′-nucleotidase
WP_265061484.1	ATP-dependent helicase HrpB
WP_265061494.1	d-hexose-6-phosphate mutarotase
WP_033136990.1	Phage tail assembly protein
WP_204481558.1	DEAD/DEAH box helicase
WP_201994931.1	Aspartate-ammonia ligase
WP_203119821.1	Acetolactate synthase 2 small subunit

靶标基因
Target gene

注释
Annotation

WP_265061909.1

2-hydroxyacid dehydrogenase

WP_201954220.1

Malate dehydrogenase

WP_005335875.1

1-acylglycerol-3-phosphate O-acyltransferase

WP_265062582.1

Penicillin-binding protein 1C

WP_265062605.1

Inositol-1-monophosphatase

WP_265061092.1

Aspartate/tyrosine/aromatic aminotransferase

WP_167566591.1

Derived by automated computational analysis using gene prediction method: protein homology.

WP_040066712.1

Nucleobase: cation symporter-2 family protein

WP_265061310.1

Bifunctional metallophosphatase/5′-nucleotidase

WP_265061484.1

ATP-dependent helicase HrpB

WP_265061494.1

d-hexose-6-phosphate mutarotase

WP_033136990.1

Phage tail assembly protein

WP_204481558.1

DEAD/DEAH box helicase

WP_201994931.1

Aspartate-ammonia ligase

WP_203119821.1

Acetolactate synthase 2 small subunit

基因编号 Gene ID	注释 Annotation	ΔssrA vs. WT (fold change)	ΔsmpB vs. WT (fold change)
WT indicates wild type, ΔssrA indicates tmRNA knockout strain, ΔsmpB indicates smpB knockout strain, −^*a−g indicates no significant difference.
WP_201994931.1	Aspartate-ammonia ligase	2.0	−^*g
WP_201954220.1	Malate dehydrogenase	−^*a	2.4
WP_265062582.1	Penicillin-binding protein 1C	−^*b	2.3
WP_265061484.1	ATP-dependent helicase HrpB	−^*c	3.5
WP_265061494.1	d-hexose-6-phosphate mutarotase	−^*d	4.7
WP_005335875.1	1-acylglycerol-3-phosphate O-acyltransferase	−^*e	3.3
WP_265061092.1	Aspartate/tyrosine/aromatic aminotransferase	−^*f	0.3

基因编号
Gene ID

注释
Annotation

ΔssrA vs. WT
(fold change)

ΔsmpB vs. WT
(fold change)

WT indicates wild type, ΔssrA indicates tmRNA knockout strain, ΔsmpB indicates smpB knockout strain, −^*a−g indicates no significant difference.

WP_201994931.1

Aspartate-ammonia ligase

2.0

−^*g

WP_201954220.1

Malate dehydrogenase

−^*a

2.4

WP_265062582.1

Penicillin-binding protein 1C

−^*b

2.3

WP_265061484.1

ATP-dependent helicase HrpB

−^*c

3.5

WP_265061494.1

d-hexose-6-phosphate mutarotase

−^*d

4.7

WP_005335875.1

1-acylglycerol-3-phosphate O-acyltransferase

−^*e

3.3

WP_265061092.1

Aspartate/tyrosine/aromatic aminotransferase

−^*f

0.3