Lon1蛋白酶参与耐辐射异常球菌高温胁迫及细胞分裂的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1067

摘要/Abstract

摘要：

Lon是一类广泛存在于生物体内的蛋白水解酶,其通过水解受损及无用蛋白,防止蛋白聚集造成的细胞伤害,在胞内氨基酸周转再利用中发挥重要作用。极端微生物耐辐射异常球菌（Deinococcus radiodurans R1）的超强非生物胁迫耐受性与其拥有功能强大的胞内蛋白质稳态系统密切相关。而Lon作为蛋白水解系统的重要组成部分,在耐辐射异常球菌中的功能分析还未见具体报导。因此本研究以耐辐射异常球菌Lon1蛋白酶为研究对象,分析其逆境胁迫下的功能。结果显示 lon1基因的转录受高温诱导,48℃高温胁迫蛋白组学分析显示,lon1的缺失使多个分子伴侣蛋白、参与膜功能和转运蛋白以及与DNA修复、转录调控、能量代谢等相关的蛋白表达量发生显著上调;lon1的缺失使5个参与细胞形态建成的蛋白表达量显著下调,电镜结果证实lon1缺失使细菌分裂异常,形成巨型细胞,细胞膜发生严重的损伤;非生物胁迫冲击实验发现,lon1的缺失使耐辐射异常球菌对0.2 mol/L NaCl以及48℃高温敏感。研究表明Lon1蛋白酶主要参与耐辐射异常球菌的细胞形态建成,并在适应高温等胁迫中发挥功能。

关键词: 耐辐射异常球菌, Lon, 非生物胁迫, 蛋白质组

Abstract:

Lon is a type of proteases that widely present in many organisms. It prevents cell damage that is caused by irreversible protein aggregation through hydrolyzing damaged and useless proteins,and plays an important role in promoting the turnover and reuse of intracellular amino acids. The supper resistance of the extremophilic microorganisms Deinococcus radiodurans R1 to abiotic stress is closely related to its powerful intracellular protein homeostasis system. However,as an important part of the proteolytic system,little is known about the role of Lon in D. radiodurans R1. Therefore,this study chosen the Lon1 of D. radiodurans as the research object to analyze its function under abiotic stress. The results showed that the transcription of lon1 was induced by high temperature. Proteomics analysis of 48℃ showed that deletion of lon1 significantly up-regulated the expression levels of several molecular chaperones,and several proteins involved in membrane function and transport,DNA repair,transcriptional regulation and energy metabolism;and deletion of lon1 down-regulated the expression levels of 5 cell morphogenesis-related proteins. Electron microscopy results confirmed that the deletion of lon1 resulted in the abnormal cell division,the formation of giant cells,and the severe damages of the cell membrane. In addition,abiotic stress experiments uncovered the deletion of lon1 made D. radiodurans sensitive to 0.2 mol/L NaCl and 48℃ high temperature. Therefore,it is speculated that Lon1 is mainly involved in the cell morphogenesis of D. radiodurans and plays a role in adapting to high temperature and other abiotic stresses.

Key words: Deinococcus radiodurans R1, Lon, abiotic stress, proteomics

赵明明, 唐殷, 郭磊周, 韩佳慧, 葛佳茗, 孟勇, 平淑珍, 周正富, 王劲. Lon1蛋白酶参与耐辐射异常球菌高温胁迫及细胞分裂的功能研究[J]. 生物技术通报, 2022, 38(5): 149-158.

ZHAO Ming-ming, TANG Yin, GUO Lei-zhou, HAN Jia-hui, GE Jia-ming, MENG Yong, PING Shu-zhen, ZHOU Zheng-fu, WANG Jin. Function Analysis of Lon1 Protease Involved in High Temperature Stress and Cell Division of Deinococcus radiodurans R1[J]. Biotechnology Bulletin, 2022, 38(5): 149-158.

图/表 9

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菌株及质粒Strains and plasmids		特性Relevant characteristic	来源Source
菌株	Deinococcus radioduransR1	耐辐射异常球菌野生型	中科院微生物所菌种保藏中心
	△lon1	缺失lon1基因的突变株（Kan^r）	本研究构建
	comlon1	lon1回补株（Kan^r+ Cm^r）	本研究构建
	Z3-△lon1	△lon1中导入质粒pRADZ3（Cm^r）	本研究构建
质粒	pRAD Z3	构建回补菌株所用质粒	本实验室保存

菌株及质粒Strains and plasmids		特性Relevant characteristic	来源Source
菌株	Deinococcus radioduransR1	耐辐射异常球菌野生型	中科院微生物所菌种保藏中心
	△lon1	缺失lon1基因的突变株（Kan^r）	本研究构建
	comlon1	lon1回补株（Kan^r+ Cm^r）	本研究构建
	Z3-△lon1	△lon1中导入质粒pRADZ3（Cm^r）	本研究构建
质粒	pRAD Z3	构建回补菌株所用质粒	本实验室保存

扩增产物 Product for PCR amplification	引物名称 Primer name	序列Sequence（5'-3'）	片段长度Size/bp
lon1-Up	lon1-UP-F	AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC	613
lon1-Up	lon1-up-R	GGGCCCTCGGTCTCCATGGCTCGGTGTCACGGGTCAGCAGCT	613
lon1-Kan	Kan-lon1-F	AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC	965
lon1-Kan	Kan-Down-R	TGGTCCACGCCAGGCCCTGCGTTAGAAAAACTCATCGAGCAT	965
lon1-Down	lon1-Down-F	ATGCTCGATGAGTTTTTCTAACGCAGGGCCTGGCGTGGACCA	614
lon1-Down	lon1-Down-R	TTATGCGCCCGGCTGCACCGA	614
Kan内部序列	YZ-Km-F	CGATTGTATGGGAAGCCCGAT	591
Kan内部序列	YZ-Km-R	CTCACCGAGGCAGTTCCATAG	591
敲除的lon1序列	YZ-lon1-F	ACCGTGGTTCGCAACTACAT	400
敲除的lon1序列	YZ-lon1-R	CGATCTCGACCTTCTCCTGC	400

扩增产物 Product for PCR amplification	引物名称 Primer name	序列Sequence（5'-3'）	片段长度Size/bp
lon1-Up	lon1-UP-F	AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC	613
lon1-Up	lon1-up-R	GGGCCCTCGGTCTCCATGGCTCGGTGTCACGGGTCAGCAGCT	613
lon1-Kan	Kan-lon1-F	AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC	965
lon1-Kan	Kan-Down-R	TGGTCCACGCCAGGCCCTGCGTTAGAAAAACTCATCGAGCAT	965
lon1-Down	lon1-Down-F	ATGCTCGATGAGTTTTTCTAACGCAGGGCCTGGCGTGGACCA	614
lon1-Down	lon1-Down-R	TTATGCGCCCGGCTGCACCGA	614
Kan内部序列	YZ-Km-F	CGATTGTATGGGAAGCCCGAT	591
Kan内部序列	YZ-Km-R	CTCACCGAGGCAGTTCCATAG	591
敲除的lon1序列	YZ-lon1-F	ACCGTGGTTCGCAACTACAT	400
敲除的lon1序列	YZ-lon1-R	CGATCTCGACCTTCTCCTGC	400

UniProtKB	Locus	Predicted function	Protein name	FC
Protein fate（heat shock responsive chaperones and protease）
Q9RV58	DR_1172	Chaperone-like protein	DosH	13.37
Q9RWQ9	DR_0607	60 kD chaperone	GroL	7.08
Q9RVI3	DR_1046	Chaperone protein ClpB	ClpB	4.70
Q9RXG4	DR_0349	Lon protease	Lon2	4.52
Q9RWR0	DR_0606	10 kD chaperone	GroS	4.46
Q9RY24	DR_0128	Chaperone protein GrpE	GrpE	3.24
DNA metabolism（repair and recombination）
Q9RXI7	DR_0326	DNA damage response protein D	DdrD	2.59
Q9RS64	DR_2263	DNA protection during starvation protein 1	Dps1	2.88
Q9RY51	DR_0099	Single-stranded DNA-binding protein	Ssb	3.25
Q9RSJ6	DR_2128	DNA-directed RNA polymerase subunit alpha	RpoA	2.06
Q9RRE4	DR_2548	Transcriptional regulatory protein		7.75
Membrane functions and transport
Q9RWU0	DR_0575	Protein translocase subunit SecA	SecA	2.18
P56867	DR_2508	Hexagonally packed intermediate-layer surface protein	Hpi	3.48
Q9RWH3	DR_0695	V-type ATP synthase subunit	AtpI	1.57
Translation
Q9RST0	DR_2043	50S ribosomal protein L7/L12	RplL	0.14
Central intermediary metabolism（synthesis of nitrogen,sulfur,polyamine compounds）
Q9RR70	DR_2627	Fumarate hydratase class II	FumC	7.59
Q9RWB2	DR_0757	Citrate synthase	GltA	3.06
Q9RZ06	DR_A0147	Histidine ammonia-lyase	HutH	2.66
Q9RTN7	DR_1720	Aconitate hydratase A	Acn	2.10
P56861	DR_A0014	Adenylyl-sulfate kinase	CysC	0.23
Amino acid biosynthesis and energy metabolism
Q9RRT0	DR_2405	Demethylmenaquinone methyltransferase	MenG	0.51
Q9RV69	DR_1161	5-hydroxyisourate hydrolase		0.33
Q9RV98	DR_1131	Ferrochelatase	HemH	2.04
Q9RRC4	DR_2568	Arginine--tRNA ligase	ArgS	0.54
Cell morphogenesis
Q9RRJ4	DR_2496	UDP-N-acetylmuramoylalanine--D-glutamate ligase	MurD	0.65
Q9RWN8	DR_0628	UDP-N-acetylenolpyruvoylglucosamine reductase	MurB	0.59
Q9RXF1	DR_0362	D-alanine--D-alanine ligase	Ddl	0.46
Q9RWN9	DR_0627	UDP-N-acetylmuramate--L-alanine ligase	MurC	0.42
Q9RXL3	DR_0297	UDP-N-acetylmuramyl-tripeptide synthetase	MurE	0.40