微生物学报

Strain or plasmid	Genotype or relevant characteristic	Source
Strains
LM201	Lm strain isolated from foodstuff^[31]	This laboratory
S0192(ΔlisK)	In-frame deletion of the lisK gene with 1 452 bp in LM201	This laboratory
S2001(ΔlisR)	In-frame deletion of the lisR gene with 681 bp in LM201	This study
S0192c(ΔlisKc)	Complementary strain of S0192(ΔlisK)	This study
S2001c(ΔlisRc)	Complementary strain of S2001(ΔlisR)	This study
DH5α/p1001	p1001 in Escherichia coli DH5α	This study
Plasmids
pHT304-ts	Temperature-sensitive shuttle plasmid	This laboratory
pHT304	The shuttle vector plasmid	This laboratory
p1001	pHT304-ts containing lisR up- and downstream homologous region of LM201	This study
plisKc	pHT304 containing 1 452 bp coding sequence of lisK gene	This study
plisRc	pHT304 containing 681 bp coding sequence of lisR gene	This study

Strain or plasmid	Genotype or relevant characteristic	Source
Strains
LM201	Lm strain isolated from foodstuff^[31]	This laboratory
S0192(ΔlisK)	In-frame deletion of the lisK gene with 1 452 bp in LM201	This laboratory
S2001(ΔlisR)	In-frame deletion of the lisR gene with 681 bp in LM201	This study
S0192c(ΔlisKc)	Complementary strain of S0192(ΔlisK)	This study
S2001c(ΔlisRc)	Complementary strain of S2001(ΔlisR)	This study
DH5α/p1001	p1001 in Escherichia coli DH5α	This study
Plasmids
pHT304-ts	Temperature-sensitive shuttle plasmid	This laboratory
pHT304	The shuttle vector plasmid	This laboratory
p1001	pHT304-ts containing lisR up- and downstream homologous region of LM201	This study
plisKc	pHT304 containing 1 452 bp coding sequence of lisK gene	This study
plisRc	pHT304 containing 681 bp coding sequence of lisR gene	This study

Primers name	Oligonucleotide sequences (3ʹ→5ʹ)	Restriction site
Mutant strain construction
lisR(R)-F	AATTCCTTTTACCCGATCAGCAGTAAACCGGATTG
lisR(R)-R	CTACTAGGCCGACATGAACTACCCATGGCGT	Kpn Ⅰ
lisR(L)-F	ATCAGGTAAATGGTCAAGGAGATCTGGC	Xba Ⅰ
lisR(L)-R	ACTGGCTTTCTCTCCCAATCCGGTTTACTGCTGAT
Complementary strain construction
lisKc-F	CCTTTTACCCGATCAGCAGTAAGATCTGGC	Xba Ⅰ
lisKc-R	GGAATGTAACTTGGTTTTAGGTTTATTCCATGGCGT	Kpn Ⅰ
lisRc-F	GTAGAAGATGCTAATTATAAGATAAGTAAGATCTGGC	Xba Ⅰ
lisRc-R	CCATACACTACGCATGTACTCCATGGCGT	Kpn Ⅰ
Primers for RT-qPCR
16S rRNA-F	AGAGTCCAGCCGATACGTAG
16S rRNA-R	ACCTGCTTTCAGACTGCCTC
pduB-F	GCAGATGTAAGCGTTAGTGATCCG
pduB-R	CCACCACTGCTACAAAGTCTACAC
flhA-F	GCAATAGTAGACTAGGCAGTT
flhA-R	GTAGCCGATAGCATGCGTTA
mngB-F	GCGAAGAACGGTAAGAAGACGA
mngB-R	GGCTAAATGAGATAGCAGGTCCAGC
flgB-F	CGTTTGAGCTCGGTTCACTA
flgB-R	CAGTTGCAGTGAAGACTTTAC
inlB-F	AGGAACTCGCTTGAATCCAGGA
inlB-R	TCACTATAACACGGTGAACGTCC
fliM-F	CTAAAGACCACTCTAACCTAC
fliM-R	GAGCCACCTTCTCTAATACTT
motA-F	TTTACCACAGGTTTCGTGC
motA-R	CCTTTTCCCTTCTTTTGCTCT
dhaL-F	TGGTTAGCGAACCAGCTCTAGTG
dhaL-R	AGTCGTTTCAACCACCGCGTA
flgC-F	CCTCGAATGAAGGTAGGTAAC
flgC-R	CAAGCTTTGTCGAAATTGTCC
rpiB-F	GTACCAGACTTCGTTACCGTTGA
rpiB-R	GGCTCTGAGCGATAATGGCAATCA
manX-F	AGTGGCACTCAACGCTCCTA
manX-R	ACCTTCGACGTTAACGTAGGAA
fliF-F	GCAGTGGTAAAACAAGGC
fliF-R	TTTTTGTGGGTCGGGAAC
flgD-F	TTCCTTTCAACGCGGT
flgD-R	TTTGAGGCGGAACGTTAG
flhF-F	GTCGATAACGGTGAAGTGAG
flhF-R	CCTTGATCGTGTTAGCTGC
fliR-F	TTTTGACCGTTGAGCCGA
fliR-R	TAAGGTCACCGTTGTACC
motB-F	GTAGCGGAAGTTCTGACG
motB-R	TCGTCGTCGTCGGAATGAA

Primers name	Oligonucleotide sequences (3ʹ→5ʹ)	Restriction site
Mutant strain construction
lisR(R)-F	AATTCCTTTTACCCGATCAGCAGTAAACCGGATTG
lisR(R)-R	CTACTAGGCCGACATGAACTACCCATGGCGT	Kpn Ⅰ
lisR(L)-F	ATCAGGTAAATGGTCAAGGAGATCTGGC	Xba Ⅰ
lisR(L)-R	ACTGGCTTTCTCTCCCAATCCGGTTTACTGCTGAT
Complementary strain construction
lisKc-F	CCTTTTACCCGATCAGCAGTAAGATCTGGC	Xba Ⅰ
lisKc-R	GGAATGTAACTTGGTTTTAGGTTTATTCCATGGCGT	Kpn Ⅰ
lisRc-F	GTAGAAGATGCTAATTATAAGATAAGTAAGATCTGGC	Xba Ⅰ
lisRc-R	CCATACACTACGCATGTACTCCATGGCGT	Kpn Ⅰ
Primers for RT-qPCR
16S rRNA-F	AGAGTCCAGCCGATACGTAG
16S rRNA-R	ACCTGCTTTCAGACTGCCTC
pduB-F	GCAGATGTAAGCGTTAGTGATCCG
pduB-R	CCACCACTGCTACAAAGTCTACAC
flhA-F	GCAATAGTAGACTAGGCAGTT
flhA-R	GTAGCCGATAGCATGCGTTA
mngB-F	GCGAAGAACGGTAAGAAGACGA
mngB-R	GGCTAAATGAGATAGCAGGTCCAGC
flgB-F	CGTTTGAGCTCGGTTCACTA
flgB-R	CAGTTGCAGTGAAGACTTTAC
inlB-F	AGGAACTCGCTTGAATCCAGGA
inlB-R	TCACTATAACACGGTGAACGTCC
fliM-F	CTAAAGACCACTCTAACCTAC
fliM-R	GAGCCACCTTCTCTAATACTT
motA-F	TTTACCACAGGTTTCGTGC
motA-R	CCTTTTCCCTTCTTTTGCTCT
dhaL-F	TGGTTAGCGAACCAGCTCTAGTG
dhaL-R	AGTCGTTTCAACCACCGCGTA
flgC-F	CCTCGAATGAAGGTAGGTAAC
flgC-R	CAAGCTTTGTCGAAATTGTCC
rpiB-F	GTACCAGACTTCGTTACCGTTGA
rpiB-R	GGCTCTGAGCGATAATGGCAATCA
manX-F	AGTGGCACTCAACGCTCCTA
manX-R	ACCTTCGACGTTAACGTAGGAA
fliF-F	GCAGTGGTAAAACAAGGC
fliF-R	TTTTTGTGGGTCGGGAAC
flgD-F	TTCCTTTCAACGCGGT
flgD-R	TTTGAGGCGGAACGTTAG
flhF-F	GTCGATAACGGTGAAGTGAG
flhF-R	CCTTGATCGTGTTAGCTGC
fliR-F	TTTTGACCGTTGAGCCGA
fliR-R	TAAGGTCACCGTTGTACC
motB-F	GTAGCGGAAGTTCTGACG
motB-R	TCGTCGTCGTCGGAATGAA

Gene_ID	Gene name	Gene description	log₂fold change	P-value	Regulate
gene0738	motA	Flagellar motor stator protein MotA	4.365 318	2.38×10^-14	Up
gene0743	flaA	MULTISPECIES: FliC/FljB family flagellin	4.144 634	7.50×10^-20	Up
gene0761	fliS	MULTISPECIES: flagellar protein FliS	4.139 050	2.22×10^-15	Up
gene0742	cheV	MULTISPECIES: chemotaxis protein	4.100 357	2.43×10^-47	Up
gene0739	motB	Flagellar motor protein MotB	3.809 529	5.06×10^-15	Up
gene0744	cheY	MULTISPECIES: response regulator	3.751 984	1.45×10^-18	Up
gene0748	-	Flagellar hook-length control protein FliK	3.729 361	5.26×10^-20	Up
gene0753	fliN	Flagellar motor switch protein	3.537 165	4.45×10^-16	Up
gene0749	flgD	MULTISPECIES: flagellar basal body rod modification protein FlgD	3.394 462	2.41×10^-14	Up
gene0760	fliD	Flagellar hook-associated protein 2	3.373 229	2.51×10^-17	Up
gene0750	flgE	Flagellar hook protein FlgE	3.358 998	2.67×10^-17	Up
gene0767	fliG	Flagellar motor switch protein FliG	3.325 708	4.46×10^-26	Up
gene0759	flgL	Flagellar hook-associated protein 3	3.300 846	2.72×10^-13	Up
gene0768	fliH	FliH/SctL family protein	3.271 808	1.22×10^-24	Up
gene0765	fliE	MULTISPECIES: flagellar hook-basal body complex protein FliE	3.271 007	2.30×10^-13	Up
gene0752	fliM	MULTISPECIES: flagellar motor switch protein FliM	3.162 440	2.96×10^-13	Up
gene0766	fliF	Flagellar basal-body MS-ring/collar protein FliF	3.151 362	3.41×10^-20	Up
gene0763	flgB	Flagellar basal body rod protein FlgB	3.074 186	7.43×10^-29	Up
gene0758	flgK	Flagellar hook-associated protein FlgK	3.042 255	2.32×10^-16	Up
gene0764	flgC	MULTISPECIES: flagellar basal body rod protein FlgC	3.021 742	1.97×10^-30	Up
gene0735	flgG	Flagellar basal-body rod protein FlgG	3.011 324	3.16×10^-8	Up
gene0769	fliI	Flagellar protein export ATPase FliI	2.750 155	1.74×10^-14	Up
gene0745	cheA	Chemotaxis protein CheA	2.697 812	1.65×10^-24	Up
gene0734	flhF	Flagellar biosynthesis protein FlhF	2.690 537	5.29×10^-8	Up
gene0733	flhA	Flagellar biosynthesis protein FlhA	2.142 607	7.07×10^-5	Up

Gene_ID	Gene name	Gene description	log₂fold change	P-value	Regulate
gene0738	motA	Flagellar motor stator protein MotA	4.365 318	2.38×10^-14	Up
gene0743	flaA	MULTISPECIES: FliC/FljB family flagellin	4.144 634	7.50×10^-20	Up
gene0761	fliS	MULTISPECIES: flagellar protein FliS	4.139 050	2.22×10^-15	Up
gene0742	cheV	MULTISPECIES: chemotaxis protein	4.100 357	2.43×10^-47	Up
gene0739	motB	Flagellar motor protein MotB	3.809 529	5.06×10^-15	Up
gene0744	cheY	MULTISPECIES: response regulator	3.751 984	1.45×10^-18	Up
gene0748	-	Flagellar hook-length control protein FliK	3.729 361	5.26×10^-20	Up
gene0753	fliN	Flagellar motor switch protein	3.537 165	4.45×10^-16	Up
gene0749	flgD	MULTISPECIES: flagellar basal body rod modification protein FlgD	3.394 462	2.41×10^-14	Up
gene0760	fliD	Flagellar hook-associated protein 2	3.373 229	2.51×10^-17	Up
gene0750	flgE	Flagellar hook protein FlgE	3.358 998	2.67×10^-17	Up
gene0767	fliG	Flagellar motor switch protein FliG	3.325 708	4.46×10^-26	Up
gene0759	flgL	Flagellar hook-associated protein 3	3.300 846	2.72×10^-13	Up
gene0768	fliH	FliH/SctL family protein	3.271 808	1.22×10^-24	Up
gene0765	fliE	MULTISPECIES: flagellar hook-basal body complex protein FliE	3.271 007	2.30×10^-13	Up
gene0752	fliM	MULTISPECIES: flagellar motor switch protein FliM	3.162 440	2.96×10^-13	Up
gene0766	fliF	Flagellar basal-body MS-ring/collar protein FliF	3.151 362	3.41×10^-20	Up
gene0763	flgB	Flagellar basal body rod protein FlgB	3.074 186	7.43×10^-29	Up
gene0758	flgK	Flagellar hook-associated protein FlgK	3.042 255	2.32×10^-16	Up
gene0764	flgC	MULTISPECIES: flagellar basal body rod protein FlgC	3.021 742	1.97×10^-30	Up
gene0735	flgG	Flagellar basal-body rod protein FlgG	3.011 324	3.16×10^-8	Up
gene0769	fliI	Flagellar protein export ATPase FliI	2.750 155	1.74×10^-14	Up
gene0745	cheA	Chemotaxis protein CheA	2.697 812	1.65×10^-24	Up
gene0734	flhF	Flagellar biosynthesis protein FlhF	2.690 537	5.29×10^-8	Up
gene0733	flhA	Flagellar biosynthesis protein FlhA	2.142 607	7.07×10^-5	Up

LisK/R通过调控鞭毛基因表达影响单增李斯特菌低温生长

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汪国俊 , 黄金梅 , 栗绍文 , 刘梅

微生物学报 | 研究报告 2025,65(7): 2889-2902

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微生物学报 | 研究报告 2025, 65(7): 2889-2902

LisK/R通过调控鞭毛基因表达影响单增李斯特菌低温生长

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汪国俊, 黄金梅, 栗绍文, 刘梅

作者信息

华中农业大学动物医学院，湖北武汉

LisK/R plays a role in Listeria monocytogenes growth at low temperatures by regulating the expression of flagellar genes

Guojun WANG, Jinmei HUANG, Shaowen LI, Mei LIU

Affiliations

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China

出版时间: 2025-07-04 doi: 10.13343/j.cnki.wsxb.20240756

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摘要

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【目的】 单增李斯特菌(Listeria monocytogenes, Lm)是一种革兰阳性食源性病原菌，可导致病死率高达20%-30%的人兽共患病——李斯特菌病(listeriosis)。Lm能够在低温下生长繁殖，对食品安全和人类健康构成极大的威胁。探究Lm低温生长机制，将为制定控制Lm低温生长的措施以及研发低温生长抑制剂提供理论依据。研究发现细菌双组分系统(two-component signal transduction system, TCS) LisK/R对Lm低温生长具有调控作用，但其具体机制尚未明确。因此，本研究旨在探讨LisK/R对Lm低温生长的作用机制。 【方法】 以Lm食品分离株LM201为亲本株，构建3个菌株：ΔlisR (LisK/R的RR缺失株)、ΔlisRc (ΔlisR的回补株)和ΔlisKc [LisK/R的HK缺失株ΔlisK (课题组之前已构建)的回补株]。测定亲本株、缺失株和回补株在4 ℃下的生长曲线；对4 ℃下生长的缺失株ΔlisK和亲本株进行RNA-Seq及数据分析；在4 ℃下，用软琼脂平板测定亲本株、缺失株和回补株的菌株运动圈直径，用透射电镜观察菌体鞭毛形成情况，用RT-qPCR测定鞭毛基因表达量。 【结果】 在4 ℃低温条件下，缺失株的生长速率显著低于亲本株(P<0.05)，而回补株的生长情况与亲本株一致。RNA-Seq基因表达差异性分析结果显示，与亲本株相比，缺失株ΔlisK的鞭毛基因表达普遍显著上调(P<0.05)。在4 ℃低温条件下，缺失株的运动圈直径显著大于亲本株和回补株(P<0.05)；电镜观察的结果显示，缺失株的鞭毛数量多，而亲本株和回补株的鞭毛数量较少；RT-qPCR测定结果也显示，缺失株的鞭毛基因表达量显著高于亲本株和回补株(P<0.05)。 【结论】 在4 ℃低温条件下，生长速率低的LisK/R缺失株鞭毛数量多，而生长速率高的亲本株鞭毛数量少，表明Lm通过LisK/R抑制鞭毛基因的表达，减少鞭毛产量，从而将能量用于生长以促进Lm在低温下的生长，其具体的分子调控机制尚需进一步研究。本研究为阐明Lm低温生长机制提供了新的信息。

关键词

单增李斯特菌 / 低温生长 / 双组分系统LisK/R / 鞭毛

Abstract

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[Objective] Listeria monocytogenes (Lm) is a Gram-positive foodborne pathogen causing human and animal listeriosis with high case fatality rates (20%-30%). Lm can grow at low temperatures, which brings great risks to food safety and human health. Exploring the mechanism of Lm growth at low temperatures will provide a theoretical basis for the formulation of measures to control Lm growth at low temperatures and the development of low-temperature growth inhibitors. Studies have shown that LisK/R, a two-component signal transduction system (TCS) in Lm, plays a role in regulating the growth of Lm at low temperatures, while the specific mechanism remains unclear. This study aims to reveal the mechanism of LisK/R in regulating the growth of Lm at low temperatures. [Methods] With the Lm strain LM201 isolated from foodstuff as the parental strain, we constructed three strains: ΔlisR (RR-deleted mutant of LisK/R), ΔlisRc (complementary strain of ΔlisR), and ΔlisKc (complementary strain of ΔlisK, which was constructed before this study in our laboratory). The growth curves of these strains were determined at 4 ℃. RNA-Seq was performed for ΔlisK and the parental strain growing at 4 ℃, and the obtained data were analyzed. We assayed the swarming area diameter on soft agar plate, flagellar biosynthesis by transmission electron microscopy, and transcriptional levels of flagellar genes by RT-qPCR for all the strains at 4 ℃. [Results] The growth of deletion mutants was slower than that of the parental strain (P<0.05), and the growth of complementary strains was consistent with that of the parental strain at 4 ℃. The RNA-Seq data showed that compared with that in the parental strain, the expression of flagellar genes in ΔlisK was up-regulated (P<0.05). At 4 ℃, the swarming area diameters of deletion mutants on soft agar plates were bigger than those of the parental strain and complementary strains (P<0.05); the deletion mutants had more flagella, while the parental and complementary strains had fewer flagella; the expression of flagellar genes in the deletion mutants was higher than that in the parental and complementary strains (P<0.05). [Conclusion] At 4 ℃, the LisK/R mutants with slow growth had more flagella, while the parental strain with rapid growth had fewer flagella. The results indicate that LisK/R inhibits the expression of flagellar genes in Lm to reduce flagellar production, and the energy is used to promote Lm growth at low temperatures. The specific molecular regulatory mechanism of LisK/R needs further study. This study provides new information for elucidating the mechanism of Lm growth at low temperatures.

Key words

Listeria monocytogenes / growth at low temperatures / two-component system LisK/R / flagellum

引用本文

汪国俊, 黄金梅, 栗绍文, 刘梅. LisK/R通过调控鞭毛基因表达影响单增李斯特菌低温生长. 微生物学报, 2025 , 65 (7) : 2889 -2902 . DOI: 10.13343/j.cnki.wsxb.20240756

Guojun WANG, Jinmei HUANG, Shaowen LI, Mei LIU. LisK/R plays a role in Listeria monocytogenes growth at low temperatures by regulating the expression of flagellar genes[J]. Acta Microbiologica Sinica, 2025 , 65 (7) : 2889 -2902 . DOI: 10.13343/j.cnki.wsxb.20240756

正文

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单核细胞增生李斯特菌(Listeria monocytogenes, Lm)简称单增李斯特菌，是一种重要的食源性病原菌，可导致人畜李斯特菌病(listeriosis)，免疫力低下者尤为易感，病死率(case fatality rate)高达20%-30%^[1-3]。Lm是一种革兰氏阳性、短杆状、不形成芽胞、兼性厌氧型细菌。Lm具有鞭毛基因，但在37 ℃时不产生鞭毛，而在25 ℃左右时有鞭毛形成^[4]。Lm能够在低温下生长^[5]，这无疑给冷藏食品带来了安全隐患，尤其是冷藏时间越长，隐患越大。因此，揭示Lm低温生长机制是防控李斯特菌病的重要基础。

各国学者从不同角度对Lm低温生长机制进行了研究。研究发现，Lm具有1个低温生长调节子LltR，其编码基因为lltR，lltR基因突变后，菌体的低温生长能力显著下降^[6-7]。Vásquez等^[8]发现，Lm低温生长离不开对钴胺素(维生素B₁₂)的生物合成。Lm在低温下可能通过多种机制适应并生长繁殖，包括改变细胞膜脂质组分以增加不饱和脂肪酸含量^[9-14]；高表达冷休克蛋白^[15-18]；合成和积累抗冻剂^[19-20]；利用rRNA甲基转移酶稳定核糖体结构和功能^[21]；通过阻遏蛋白MogR减少鞭毛的产生^[22]等。此外，双组分系统的调控也是Lm低温生长的关键因素^[23-26]。

双组分系统(two-component signal transduction system, TCS)是细菌响应温度变化等环境因素的重要调控系统^[27-28]。一个细菌可以拥有多个TCS，一个典型的TCS由膜结合感应激酶(membrane-bound sensory kinase)，即组氨酸激酶(histidine kinase, HK)和胞质中应答调节子(response regulator, RR)组成，少数TCS不包含HK组分，仅包含RR组分，称为孤儿RR^[29]。HK感知胞外信号后发生自体磷酸化，随后将磷酸基团转移给配对的RR，使其活化。活化的RR作为转录因子结合到靶基因启动子序列上，对靶基因的表达进行上调或下调，从而开启菌体应对新环境的生存模式^[30]。

本课题组以从食品中分离的一株血清型为4b的Lm分离株(命名为LM201)为研究材料，通过对LM201全基因组测序^[31]和TCS分析发现，LM201拥有14个TCS (HK/RR)和2个孤儿RR^[32]。除必需HK基因yycG外，本课题组构建了13个TCS的HK缺失株并测定了这些缺失株在低温4 ℃下的生长情况，发现与37 ℃相比，HK基因lisK缺失株ΔlisK的生长速率显著下降。Chan等^[23-24]发现，与37 ℃相比，在4 ℃下Lm菌株10403S的lisK表达水平显著上调，而与之配对的RR基因lisR缺失株ΔlisR在4 ℃下生长受限，但在37 ℃时能正常生长。同样地，与37 ℃相比，在3 ℃下Lm菌株EGD-e的lisK表达水平显著上调，而当缺失lisK后，菌株在3 ℃下生长受限，但在37 ℃时与亲本株无差异^[25]。上述研究表明，LisK/R这对TCS对Lm低温生长具有调控作用，但其具体机制尚不明确。鉴于此，本研究以LisK/R为切入点，探寻影响Lm低温生长的因素，从而为解析Lm低温生长机制提供新的信息。

1 材料与方法

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1.1 菌株、质粒和引物

表1详细列出了本研究所用的菌株与质粒信息。本研究构建缺失株和回补株以及用于RT-qPCR的引物见表2，引物均由武汉金开瑞生物工程有限公司合成。

1.2 主要试剂和仪器

BHI培养基，BD公司；氨苄青霉素(ampicillin, AMP)、红霉素(erythromycin, ERY)，武汉华大至善生物科技有限公司；电镜负染液磷钨酸(phosphotungstic acid, PTA)，中国科学院武汉病毒研究所；高保真DNA聚合酶、反转录试剂盒，南京诺唯赞生物科技股份有限公司；SYBR qPCR Master Mix，武汉擎科生物科技有限公司；凝胶回收试剂盒和质粒提取试剂盒，Omega Bio-Tek公司；T4 DNA连接酶、限制性内切酶，宝生物工程(大连)有限公司(TaKaRa公司)。

超净工作台，哈尔滨东联电子技术开发有限公司；PCR仪、荧光定量PCR仪和凝胶成像系统，Bio-Rad公司；核酸电泳仪，北京六一生物科技有限公司；紫外-可见分光光度计、100 kV透射电镜和冷冻高速离心机，Hitachi公司；电热恒温水浴锅，北京市长风仪器仪表公司；全温度振荡培养箱(摇床)，苏州培英实验室设备有限公司；水平离心机，上海安亭科学仪器厂；小型高速离心机，Eppendorf公司；4 ℃和-30 ℃冰箱，Haier公司。

1.3 缺失株和回补株的构建

依照汪国俊等^[22]的方法，以Lm菌株LM201的DNA为模板，利用表2中的引物lisR(L)-F/R和lisR(R)-F/R对lisR的上、下游同源臂进行PCR扩增。利用Overlap PCR将扩增获得的上、下游同源臂进行连接，将连接产物克隆至温敏型自杀载体pHT304-ts，从而得到携带lisR同源臂的重组质粒p1001。将该重组质粒转化至Escherichia coli DH5α感受态细胞，获得重组菌株DH5α/p1001。对p1001中的同源臂序列进行验证性测序(武汉天一辉远生物技术公司)，测序结果符合预期。将纯化的p1001质粒通过电转化法(参数2.4 kV)导入LM201感受态细胞，经筛选获得阳性转化子后，利用同源重组技术成功构建lisR缺失株S2001(ΔlisR)。为构建回补菌株，用引物lisKc-F/R (表2)扩增lisK基因片段，并将其插入穿梭载体pHT304，获得重组质粒plisKc。经测序验证正确的plisKc质粒通过电转导入缺失株S0192 (ΔlisK)感受态细胞，最终通过PCR鉴定获得回补菌株S0192c(ΔlisKc)。值得注意的是，重组质粒plisRc和回补株S2001c(ΔlisRc)的构建流程与上述过程基本一致，仅将扩增引物更换为lisRc-F/R (表2)。

1.4 生长曲线测定

依照汪国俊等^[22]的方法，挑取LM201、S0192(ΔlisK)、S2001(ΔlisR)、S0192c(ΔlisKc)和S2001c(ΔlisRc)单菌落，接种于BHI液体培养基，在37 ℃、200 r/min条件下活化培养12 h，然后按1:100转接至新鲜BHI，在37 ℃、200 r/min条件下培养至OD₆₀₀值为1.0，再次以相同接种量转接至新鲜BHI用于4 ℃和37 ℃条件下的菌株生长曲线测定：菌液于4 ℃恒温环境中静置培养，每隔1 d测定OD₆₀₀值，连续培养15 d；菌液于37 ℃、200 r/min振荡培养，每隔1 h测定OD₆₀₀值，连续培养12 h。每个菌株设3个重复，实验重复3次。使用GraphPad Prism 8对数据进行统计分析和绘制生长曲线图。

1.5 RNA-Seq及验证

采用TRIzol法提取4 ℃静置培养10 d (低温对数期)的S0192(ΔlisK)和LM201的总RNA，RNA的纯度和浓度采用NanoDrop 2000c进行检测，RNA的完整性通过1%琼脂糖凝胶电泳进行检测。将检测合格的样品送至武汉博越致和生物科技有限公司进行转录组测序(RNA-Seq)。使用Hisat2软件将测序获得的clean reads比对到参考基因组(https://www.ncbi.nlm.nih.gov/nuccore/CP031141.1)，并使用R语言包DESeq2分析差异表达基因，筛选差异表达基因(differentially expressed genes, DEGs)的阈值为P-value<0.05且|log₂ fold change|>1。

以16S rRNA为内参基因^[33]，按照SYBR qPCR Mix说明书(武汉擎科生物科技有限公司)，对随机挑选的RNA-Seq差异基因rpiB、dhaL、pduB、mngB、manX、inlB、flhA和motA进行RT-qPCR验证，采用2^-ΔΔ^C^t相对定量法对结果进行表达差异性分析，以验证RNA-Seq数据的可靠性。

1.6 菌株运动性测定

参照Cheng等^[34]描述的方法，用0.25%琼脂含量的BHI软平板进行菌株运动性测定。挑取菌株LM201、S0192(ΔlisK)、S2001(ΔlisR)、S0192c(ΔlisKc)和S2001c(ΔlisRc)单菌落，接种于BHI液体培养基进行活化培养，37 ℃、200 r/min培养12 h后，以1:100接种量转接至新鲜BHI培养基继续培养，直至菌液OD₆₀₀值达到1.0，获得运动性测定的菌悬液。取5 μL菌悬液，穿刺接种于0.25%琼脂含量的BHI软平板，分别置于4 ℃培养8 d和37 ℃培养48 h，然后测量平板接种点菌体生长圈直径。每个菌株设3个重复，实验重复2次。实验数据通过GraphPad Prism 8软件进行统计学分析及图形化。

1.7 透射电子显微镜观察

依照汪国俊等^[22]的方法，将LM201、S2001(ΔlisR)、S0192(ΔlisK)、S2001c(ΔlisRc)和S0192c(ΔlisKc)分别在4 ℃静置培养10 d (低温对数期)和37 ℃、200 r/min培养8 h (适温对数期)，收集菌液1 mL，置于1.5 mL EP管中，以2 000 r/min离心10 min。弃上清后，用1 mL无菌1×PBS缓冲液充分洗涤菌体沉淀，然后用同体积PBS重悬菌体，重悬的菌体即可作为透射电子显微镜(transmission electron microscope, TEM)的观察样品。在预冷的封口膜表面依次滴加3个液滴，分别为20 μL样品、超纯水和PTA染液。将铜网首先浸入样品液滴，吸附约1 min，用滤纸吸除多余液体；然后转移至超纯水液滴进行漂洗，吸干；最后浸入PTA染液液滴，负染约1 min，吸除多余染液。铜网置于室温干燥后，通过TEM观察细菌鞭毛形成情况。

1.8 鞭毛基因转录组RT-qPCR测定

依照汪国俊等^[22]的方法，收集4 ℃静置培养10 d (低温对数期)的LM201、S0192(ΔlisK)、S2001(ΔlisR)、S0192c(ΔlisKc)和S2001c(ΔlisRc)菌体，提取RNA，RNA的纯度和浓度采用NanoDrop 2000c进行检测，RNA的完整性通过1%琼脂糖凝胶电泳进行检测。用反转录试剂盒将总RNA逆转录为cDNA，作为RT-qPCR的模板。按照SYBR qPCR Mix的标准化流程，以16S rRNA为内参基因，检测鞭毛合成基因flgB、flgC、flgD、fliF、fliM、fliR、flhA、flhF、motA和motB的转录水平。采用2^-ΔΔ^C^t相对定量法对结果进行表达差异性分析。

1.9 数据统计分析

实验数据以mean±SD表示，使用双尾t检验进行差异显著性分析，*表示P<0.05 (差异显著)；**表示P<0.01 (差异非常显著)；***表示P<0.001 (差异极其显著)。

2 结果与分析

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2.1 低温4 ℃下缺失株生长情况

将亲本株(LM201)、2个缺失株[S0192(ΔlisK)和S2001(ΔlisR)]以及2个回补株[S0192c(ΔlisKc)和S2001c(ΔlisRc)])分别在4 ℃和37 ℃下培养，用测定的菌液OD₆₀₀值绘制出每个菌的生长曲线。在4 ℃生长条件下，2个缺失株在对数期及稳定期的生长速率显著低于亲本株(P<0.05或P<0.01)，而2个回补株的生长情况与亲本株保持一致(图1A)。在37 ℃生长条件下，亲本株、缺失株和回补株的生长表型未见显著差异(图1B)。结果表明，双组分系统LisK/R对Lm的低温生长具有调控作用。

2.2 缺失株S0192(ΔlisK)与亲本株LM201在4 ℃下生长的转录组测序分析

为了探明低温4 ℃下缺失株生长速率显著下降的原因，本研究对4 ℃条件下生长的缺失株S0192(ΔlisK)和亲本株LM201进行了转录组测序(RNA-Seq)，并对测序结果通过RT-qPCR进行验证。结果显示，测序结果与RT-qPCR检测结果的相关系数为0.816 7 (图2)，表明转录组测序结果可信。

分析RNA-Seq结果发现，与亲本株相比，缺失株的鞭毛合成基因表达普遍显著上调(P<0.05) (图3、表3)，这提示在低温4 ℃条件下，LisK/R这对双组分系统对鞭毛基因的转录表达具有负向调控作用。

2.3 低温4 ℃下缺失株的运动情况、鞭毛形成情况和鞭毛基因表达情况

为了验证在4 ℃生长条件下双组分系统LisK/R对鞭毛合成基因表达具有负向调控作用这一转录组测序结果分析的推测，本研究测定了亲本株(LM201)、2个缺失株[S0192(ΔlisK)和S2001(ΔlisR)]以及2个回补株[S0192c(ΔlisKc)和S2001c(ΔlisRc)]在4 ℃生长条件下的运动性、鞭毛形成情况和鞭毛基因表达量。

2.3.1 菌株的运动性

将菌株接种于0.25%琼脂含量的BHI软平板上，分别置于4 ℃培养8 d和37 ℃培养48 h，通过测量细菌的生长圈直径大小来判断菌株的运动性。结果显示，在4 ℃培养条件下，缺失株的生长圈直径显著大于亲本株(P<0.05)，而回补株的生长圈直径与亲本株无显著差异；在37 ℃培养条件下，亲本株、缺失株和回补株之间无显著差异(图4、图5)。结果表明，当菌株处于4 ℃低温时，LisK/R缺失株的运动性显著强于亲本株。

2.3.2 透射电镜(TEM)观察菌体鞭毛形成情况

将制备的4 ℃ (10 d)和37 ℃ (8 h)培养条件下的菌体样品，通过TEM观察菌体鞭毛形成情况。镜检结果显示，在4 ℃条件下，2个缺失株均呈现出密集的周身鞭毛，其鞭毛数量远多于亲本株；回补株S0192c(ΔlisKc)未见鞭毛，回补株S2001c(ΔlisRc)仅有少量鞭毛(图6)。在37 ℃条件下，所有菌株均未见鞭毛生长(图6)。结果表明，在低温4 ℃时，LisK/R缺失株的鞭毛形成量明显多于亲本株。

2.3.3 RT-qPCR测定鞭毛基因表达量

制备4 ℃条件下培养至对数期(10 d)菌体的RNA，通过RT-qPCR测定鞭毛基因转录表达量。结果显示，与亲本株相比，2个缺失株的鞭毛基因flgB、flgD、fliF、fliM、fliR、motA和motB表达均显著上调(P<0.05)；与缺失株相比，对应的2个回补株的这些鞭毛基因表达显著下调(图7、图8)。结果表明，在低温4 ℃时，LisK/R缺失株的鞭毛基因转录本表达量显著高于亲本株。

综上所述，在低温4 ℃时，LisK/R缺失株的鞭毛基因转录本表达量高于亲本株，导致缺失株的鞭毛数量多于亲本株，进而使其运动性强于亲本株。这3个结果进一步印证了转录组测序的分析结果。

3 讨论

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3.1 TCS对细菌鞭毛形成的调控作用

不同细菌的TCS对鞭毛形成的调控方式各异，有的是正向调控，即激活鞭毛基因的表达，例如空肠弯曲菌(Campylobacter jejuni)的FlgS/R^[35]；而有的则是负向调控，即抑制鞭毛的形成，例如支气管败血波氏菌(Bordetella bronchiseptica)的BvgS/A^[36]。在本研究中，4 ℃低温条件下，LisK/R缺失株的运动能力显著强于亲本株(图4、图5)，电镜观察显示缺失株的鞭毛数量多于亲本株(图6)，且缺失株的鞭毛基因转录本表达量显著高于亲本株(图7、图8)，表明LisK/R在4 ℃低温下对Lm的鞭毛形成具有抑制作用，其具体的分子机制正在探究中。

3.2 Lm 鞭毛形成的温度依赖性

鞭毛是细菌的运动器官。Lm具有鞭毛基因，但在37 ℃时不产生鞭毛，仅在30 ℃及以下时才产生鞭毛^[4]，这一特性与Lm既是腐生菌又是胞内寄生菌的特性密切相关^[37]。在37 ℃下，对应于宿主的生理环境，若产生鞭毛则不利于菌体在宿主体内的生存，因为鞭毛会被宿主免疫细胞的模式识别受体识别，从而刺激宿主清除细菌^[38]，因此，Lm在37 ℃时抑制鞭毛基因的表达；在30 ℃下，对应于腐生环境，鞭毛基因的抑制被解除，菌体产生鞭毛，便于细菌寻找营养^[39]。然而，Durack等^[40]发现，在4 ℃低温下Lm的鞭毛基因表达下调，运动性降低。本课题组的研究也显示，在4 ℃低温下Lm菌株ATCC 19115的运动性、鞭毛产量和鞭毛基因表达量均显著低于28 ℃时的水平^[22]，表明低温下Lm的鞭毛基因表达受到抑制。本研究结果进一步表明，在4 ℃低温下鞭毛基因表达受到双组分系统LisK/R的抑制。

3.3 鞭毛形成与 Lm 低温生长的关系

为什么Lm在低温下会抑制鞭毛基因的表达以减少鞭毛产量？鞭毛的合成和运动是细菌在细胞外环境中的一种有益生存机制，细菌通过鞭毛运动向最佳生长环境移动，反之则从不利环境中撤出，然而鞭毛的合成和运动对能量的需求极高^[41]。Wösten等^[35]发现，无鞭毛的空肠弯曲菌FlgS/R突变株在肉汤培养基中的生长速率比野生株快3倍。本研究结果也显示，在4 ℃低温下鞭毛多的LisK/R缺失株S0192和S2001在BHI液体培养基中的生长速率显著低于鞭毛少的亲本株(图1、图6)，这提示细菌可能采取了通过抑制鞭毛基因表达以减少鞭毛产量，从而将有限的能量用于生长繁殖的生存策略。

3.4 低温下LisK缺失后除鞭毛基因外其他基因的表达变化

本研究分析了4 ℃下生长的缺失株ΔlisK和亲本株的转录组测序结果，发现与亲本株相比，除鞭毛基因显著上调外，ΔlisK中涉及磷酸转移酶系统(phosphotransferase system, PTS)的基因有88.6% (31/35)发生下调，其中manXa基因(编码PTS系统中的甘露糖转运蛋白EIIA)下调极其显著(log₂ fold change为-5.14)。另外，murR基因(属于MurR/RpiR家族转录调控因子)也显著下调(log₂ fold change为-1.67)。MurR参与细菌细胞壁肽聚糖的再循环，调控肽聚糖中N-乙酰胞壁酸(N-acetylmuramic acid, MurNAc)的分解代谢^[42]。这提示在低温下MurNAc的分解代谢可能在LisK/R的调控下对Lm的低温生长发挥作用，相关研究尚未见报道，本课题组正在对此进行深入研究。

4 结论

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本研究表明，在4 ℃低温条件下生长速率较低的LisK/R缺失株鞭毛数量较多，而生长速率较高的亲本株鞭毛数量较少。鞭毛基因的表达及其组装过程需要消耗大量能量。在低温环境下通过降低鞭毛基因的表达、减少鞭毛的生成，并将节省的能量用于生长，是Lm采取的一种适应低温的生长策略。本研究结果进一步证实，双组分系统LisK/R通过抑制鞭毛基因的表达，促进了Lm在低温条件下的生长。然而，其具体的分子调控机制仍有待进一步研究。

作者利益冲突公开声明

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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。

基金

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国家重点研发计划(2022YFD1800100)

参考文献

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2025年第65卷第7期

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doi: 10.13343/j.cnki.wsxb.20240756

接收时间：2024-11-27
首发时间：2026-02-06
出版时间：2025-07-04

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收稿日期：2024-11-27
录用日期：2025-02-05

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National Key Research and Development Program of China(2022YFD1800100)

国家重点研发计划(2022YFD1800100)

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华中农业大学动物医学院，湖北武汉

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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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Strain or plasmid	Genotype or relevant characteristic	Source
Strains
LM201	Lm strain isolated from foodstuff^[31]	This laboratory
S0192(ΔlisK)	In-frame deletion of the lisK gene with 1 452 bp in LM201	This laboratory
S2001(ΔlisR)	In-frame deletion of the lisR gene with 681 bp in LM201	This study
S0192c(ΔlisKc)	Complementary strain of S0192(ΔlisK)	This study
S2001c(ΔlisRc)	Complementary strain of S2001(ΔlisR)	This study
DH5α/p1001	p1001 in Escherichia coli DH5α	This study
Plasmids
pHT304-ts	Temperature-sensitive shuttle plasmid	This laboratory
pHT304	The shuttle vector plasmid	This laboratory
p1001	pHT304-ts containing lisR up- and downstream homologous region of LM201	This study
plisKc	pHT304 containing 1 452 bp coding sequence of lisK gene	This study
plisRc	pHT304 containing 681 bp coding sequence of lisR gene	This study

Strain or plasmid

Genotype or relevant characteristic

Source

Strains

LM201

Lm strain isolated from foodstuff^[31]

This laboratory

S0192(ΔlisK)

In-frame deletion of the lisK gene with 1 452 bp in LM201

This laboratory

S2001(ΔlisR)

In-frame deletion of the lisR gene with 681 bp in LM201

This study

S0192c(ΔlisKc)

Complementary strain of S0192(ΔlisK)

This study

S2001c(ΔlisRc)

Complementary strain of S2001(ΔlisR)

This study

DH5α/p1001

p1001 in Escherichia coli DH5α

This study

Plasmids

pHT304-ts

Temperature-sensitive shuttle plasmid

This laboratory

pHT304

The shuttle vector plasmid

This laboratory

p1001

pHT304-ts containing lisR up- and downstream homologous region of LM201

This study

plisKc

pHT304 containing 1 452 bp coding sequence of lisK gene

This study

plisRc

pHT304 containing 681 bp coding sequence of lisR gene

This study

Primers name	Oligonucleotide sequences (3ʹ→5ʹ)	Restriction site
Mutant strain construction
lisR(R)-F	AATTCCTTTTACCCGATCAGCAGTAAACCGGATTG
lisR(R)-R	CTACTAGGCCGACATGAACTACCCATGGCGT	Kpn Ⅰ
lisR(L)-F	ATCAGGTAAATGGTCAAGGAGATCTGGC	Xba Ⅰ
lisR(L)-R	ACTGGCTTTCTCTCCCAATCCGGTTTACTGCTGAT
Complementary strain construction
lisKc-F	CCTTTTACCCGATCAGCAGTAAGATCTGGC	Xba Ⅰ
lisKc-R	GGAATGTAACTTGGTTTTAGGTTTATTCCATGGCGT	Kpn Ⅰ
lisRc-F	GTAGAAGATGCTAATTATAAGATAAGTAAGATCTGGC	Xba Ⅰ
lisRc-R	CCATACACTACGCATGTACTCCATGGCGT	Kpn Ⅰ
Primers for RT-qPCR
16S rRNA-F	AGAGTCCAGCCGATACGTAG
16S rRNA-R	ACCTGCTTTCAGACTGCCTC
pduB-F	GCAGATGTAAGCGTTAGTGATCCG
pduB-R	CCACCACTGCTACAAAGTCTACAC
flhA-F	GCAATAGTAGACTAGGCAGTT
flhA-R	GTAGCCGATAGCATGCGTTA
mngB-F	GCGAAGAACGGTAAGAAGACGA
mngB-R	GGCTAAATGAGATAGCAGGTCCAGC
flgB-F	CGTTTGAGCTCGGTTCACTA
flgB-R	CAGTTGCAGTGAAGACTTTAC
inlB-F	AGGAACTCGCTTGAATCCAGGA
inlB-R	TCACTATAACACGGTGAACGTCC
fliM-F	CTAAAGACCACTCTAACCTAC
fliM-R	GAGCCACCTTCTCTAATACTT
motA-F	TTTACCACAGGTTTCGTGC
motA-R	CCTTTTCCCTTCTTTTGCTCT
dhaL-F	TGGTTAGCGAACCAGCTCTAGTG
dhaL-R	AGTCGTTTCAACCACCGCGTA
flgC-F	CCTCGAATGAAGGTAGGTAAC
flgC-R	CAAGCTTTGTCGAAATTGTCC
rpiB-F	GTACCAGACTTCGTTACCGTTGA
rpiB-R	GGCTCTGAGCGATAATGGCAATCA
manX-F	AGTGGCACTCAACGCTCCTA
manX-R	ACCTTCGACGTTAACGTAGGAA
fliF-F	GCAGTGGTAAAACAAGGC
fliF-R	TTTTTGTGGGTCGGGAAC
flgD-F	TTCCTTTCAACGCGGT
flgD-R	TTTGAGGCGGAACGTTAG
flhF-F	GTCGATAACGGTGAAGTGAG
flhF-R	CCTTGATCGTGTTAGCTGC
fliR-F	TTTTGACCGTTGAGCCGA
fliR-R	TAAGGTCACCGTTGTACC
motB-F	GTAGCGGAAGTTCTGACG
motB-R	TCGTCGTCGTCGGAATGAA

Primers name

Oligonucleotide sequences (3ʹ→5ʹ)

Restriction site

Mutant strain construction

lisR(R)-F

AATTCCTTTTACCCGATCAGCAGTAAACCGGATTG

lisR(R)-R

CTACTAGGCCGACATGAACTACCCATGGCGT

Kpn Ⅰ

lisR(L)-F

ATCAGGTAAATGGTCAAGGAGATCTGGC

Xba Ⅰ

lisR(L)-R

ACTGGCTTTCTCTCCCAATCCGGTTTACTGCTGAT

Complementary strain construction

lisKc-F

CCTTTTACCCGATCAGCAGTAAGATCTGGC

Xba Ⅰ

lisKc-R

GGAATGTAACTTGGTTTTAGGTTTATTCCATGGCGT

Kpn Ⅰ

lisRc-F

GTAGAAGATGCTAATTATAAGATAAGTAAGATCTGGC

Xba Ⅰ

lisRc-R

CCATACACTACGCATGTACTCCATGGCGT

Kpn Ⅰ

Primers for RT-qPCR

16S rRNA-F

AGAGTCCAGCCGATACGTAG

16S rRNA-R

ACCTGCTTTCAGACTGCCTC

pduB-F

GCAGATGTAAGCGTTAGTGATCCG

pduB-R

CCACCACTGCTACAAAGTCTACAC

flhA-F

GCAATAGTAGACTAGGCAGTT

flhA-R

GTAGCCGATAGCATGCGTTA

mngB-F

GCGAAGAACGGTAAGAAGACGA

mngB-R

GGCTAAATGAGATAGCAGGTCCAGC

flgB-F

CGTTTGAGCTCGGTTCACTA

flgB-R

CAGTTGCAGTGAAGACTTTAC

inlB-F

AGGAACTCGCTTGAATCCAGGA

inlB-R

TCACTATAACACGGTGAACGTCC

fliM-F

CTAAAGACCACTCTAACCTAC

fliM-R

GAGCCACCTTCTCTAATACTT

motA-F

TTTACCACAGGTTTCGTGC

motA-R

CCTTTTCCCTTCTTTTGCTCT

dhaL-F

TGGTTAGCGAACCAGCTCTAGTG

dhaL-R

AGTCGTTTCAACCACCGCGTA

flgC-F

CCTCGAATGAAGGTAGGTAAC

flgC-R

CAAGCTTTGTCGAAATTGTCC

rpiB-F

GTACCAGACTTCGTTACCGTTGA

rpiB-R

GGCTCTGAGCGATAATGGCAATCA

manX-F

AGTGGCACTCAACGCTCCTA

manX-R

ACCTTCGACGTTAACGTAGGAA

fliF-F

GCAGTGGTAAAACAAGGC

fliF-R

TTTTTGTGGGTCGGGAAC

flgD-F

TTCCTTTCAACGCGGT

flgD-R

TTTGAGGCGGAACGTTAG

flhF-F

GTCGATAACGGTGAAGTGAG

flhF-R

CCTTGATCGTGTTAGCTGC

fliR-F

TTTTGACCGTTGAGCCGA

fliR-R

TAAGGTCACCGTTGTACC

motB-F

GTAGCGGAAGTTCTGACG

motB-R

TCGTCGTCGTCGGAATGAA

Gene_ID	Gene name	Gene description	log₂fold change	P-value	Regulate
gene0738	motA	Flagellar motor stator protein MotA	4.365 318	2.38×10^-14	Up
gene0743	flaA	MULTISPECIES: FliC/FljB family flagellin	4.144 634	7.50×10^-20	Up
gene0761	fliS	MULTISPECIES: flagellar protein FliS	4.139 050	2.22×10^-15	Up
gene0742	cheV	MULTISPECIES: chemotaxis protein	4.100 357	2.43×10^-47	Up
gene0739	motB	Flagellar motor protein MotB	3.809 529	5.06×10^-15	Up
gene0744	cheY	MULTISPECIES: response regulator	3.751 984	1.45×10^-18	Up
gene0748	-	Flagellar hook-length control protein FliK	3.729 361	5.26×10^-20	Up
gene0753	fliN	Flagellar motor switch protein	3.537 165	4.45×10^-16	Up
gene0749	flgD	MULTISPECIES: flagellar basal body rod modification protein FlgD	3.394 462	2.41×10^-14	Up
gene0760	fliD	Flagellar hook-associated protein 2	3.373 229	2.51×10^-17	Up
gene0750	flgE	Flagellar hook protein FlgE	3.358 998	2.67×10^-17	Up
gene0767	fliG	Flagellar motor switch protein FliG	3.325 708	4.46×10^-26	Up
gene0759	flgL	Flagellar hook-associated protein 3	3.300 846	2.72×10^-13	Up
gene0768	fliH	FliH/SctL family protein	3.271 808	1.22×10^-24	Up
gene0765	fliE	MULTISPECIES: flagellar hook-basal body complex protein FliE	3.271 007	2.30×10^-13	Up
gene0752	fliM	MULTISPECIES: flagellar motor switch protein FliM	3.162 440	2.96×10^-13	Up
gene0766	fliF	Flagellar basal-body MS-ring/collar protein FliF	3.151 362	3.41×10^-20	Up
gene0763	flgB	Flagellar basal body rod protein FlgB	3.074 186	7.43×10^-29	Up
gene0758	flgK	Flagellar hook-associated protein FlgK	3.042 255	2.32×10^-16	Up
gene0764	flgC	MULTISPECIES: flagellar basal body rod protein FlgC	3.021 742	1.97×10^-30	Up
gene0735	flgG	Flagellar basal-body rod protein FlgG	3.011 324	3.16×10^-8	Up
gene0769	fliI	Flagellar protein export ATPase FliI	2.750 155	1.74×10^-14	Up
gene0745	cheA	Chemotaxis protein CheA	2.697 812	1.65×10^-24	Up
gene0734	flhF	Flagellar biosynthesis protein FlhF	2.690 537	5.29×10^-8	Up
gene0733	flhA	Flagellar biosynthesis protein FlhA	2.142 607	7.07×10^-5	Up

Gene_ID

Gene name

Gene description

log₂fold change

P-value

Regulate

gene0738

motA

Flagellar motor stator protein MotA

4.365 318

2.38×10^-14

gene0743

flaA

MULTISPECIES: FliC/FljB family flagellin

4.144 634

7.50×10^-20

gene0761

fliS

MULTISPECIES: flagellar protein FliS

4.139 050

2.22×10^-15

gene0742

cheV

MULTISPECIES: chemotaxis protein

4.100 357

2.43×10^-47

gene0739

motB

Flagellar motor protein MotB

3.809 529

5.06×10^-15

gene0744

cheY

MULTISPECIES: response regulator

3.751 984

1.45×10^-18

gene0748

Flagellar hook-length control protein FliK

3.729 361

5.26×10^-20

gene0753

fliN

Flagellar motor switch protein

3.537 165

4.45×10^-16

gene0749

flgD

MULTISPECIES: flagellar basal body rod modification protein FlgD

3.394 462

2.41×10^-14

gene0760

fliD

Flagellar hook-associated protein 2

3.373 229

2.51×10^-17

gene0750

flgE

Flagellar hook protein FlgE

3.358 998

2.67×10^-17

gene0767

fliG

Flagellar motor switch protein FliG

3.325 708

4.46×10^-26

gene0759

flgL

Flagellar hook-associated protein 3

3.300 846

2.72×10^-13

gene0768

fliH

FliH/SctL family protein

3.271 808

1.22×10^-24

gene0765

fliE

MULTISPECIES: flagellar hook-basal body complex protein FliE

3.271 007

2.30×10^-13

gene0752

fliM

MULTISPECIES: flagellar motor switch protein FliM

3.162 440

2.96×10^-13

gene0766

fliF

Flagellar basal-body MS-ring/collar protein FliF

3.151 362

3.41×10^-20

gene0763

flgB

Flagellar basal body rod protein FlgB

3.074 186

7.43×10^-29

gene0758

flgK

Flagellar hook-associated protein FlgK

3.042 255

2.32×10^-16

gene0764

flgC

MULTISPECIES: flagellar basal body rod protein FlgC

3.021 742

1.97×10^-30

gene0735

flgG

Flagellar basal-body rod protein FlgG

3.011 324

3.16×10^-8

gene0769

fliI

Flagellar protein export ATPase FliI

2.750 155

1.74×10^-14

gene0745

cheA

Chemotaxis protein CheA

2.697 812

1.65×10^-24

gene0734

flhF

Flagellar biosynthesis protein FlhF

2.690 537

5.29×10^-8

gene0733

flhA

Flagellar biosynthesis protein FlhA

2.142 607

7.07×10^-5