Preliminary study on the gene function of a novel toxin-antitoxin system MSMEG_3435-3436 in Mycobacterium smegmatis

doi:10.3969/j.issn.1000-6621.2020.02.010

Abstract

Abstract:

Objective To investigate the function of the toxin-antitoxin (TA) systems in Mycobacterium smegmatis (M. smegmatis) and its role in bacterial drug tolerance. Methods Two toxin genes (MSMEG_3436 and MSMEG_6760) were constructed in anhydrous tetracycline (ATc)-induced shuttle plasmid, respectively, and then tested for repression of cell growth in M.smegmatis. The ΔMSMEG_3435-3436 and ΔMSMEG_6762-6760 mutants were constructed using CRISPR-Cas12a genome editing system to investigate the effect of TA system on cell growth. Wild type M.smegmatis and MSMEG_3435-3436 deletion mutant were treated with isoniazid (96 μg/ml) and rifampicin (40 μg/ml) and tested for drug tolerance by calculating survival rate. The TA genes (MSMEG_1277-1278, MSMEG_1283-1284, MSMEG_3435-3436, MSMEG_4447-4448, MSMEG_5635-5634) was replaced by LacZ reporter gene in M.smegmatis, resulting in promoter activity analysis stains SY3328, SY3309, SY6407, SY3310, and SY3311. The empty vector pMV261 and its derivatives expressing antitoxins were transformed into the aforementioned strains, respectively. The promoter activity of TA genes was then assessed by measurement of β-galactosidase. Results Highly expression of toxin gene MSMEG_3436 but not MSMEG_6760 in M.smegmatis repressed cell growth, while co-expression of anti-toxin gene MSMEG_3435 relieved the repression. The wild-type, ΔMSMEG_3435-3436 and ΔMSMEG_6762-6760 mutants showed similar growth phenotype in 7H9 liquid medium. Wild-type strain and ΔMSMEG_3435-3436 mutant treated with isoniazid and rifampicin had similar survival rate ((4.38±1.48) % and (3.49±0.66) %, (0.15±0.04) % and (0.03±0.02) %), which suggested that MSMEG_3435-3436 might not play an important role in drug tolerance of M.smegmatis (t=0.548, P=0.613; t=2.663, P=0.056). Promoter activity analysis showed that the β-galactosidase activities ((376.50±17.13) and (315.50±20.71) Miller units (MU), (189.00±12.24) and (160.70±9.89) MU, (225.20±9.95) and (211.70±2.57) MU, (221.40±12.07) and (186.60±13.17) MU, (179.10±5.87) and (127.70±19.21) MU) in the reporter strains harboring empty vector and antitoxin expressing plasmids were not significantly different (t=2.272, P=0.086; t=1.795, P=0.147; t=1.319, P=0.258; t=1.949, P=0.123; t=2.562, P=0.063). Conclusion The MSMEG_3435-3436 and MSMEG_6762-6760 induced expression systems and knockout strains are constructed in M.smegmatis. Further study shows that MSMEG_3435-3436 is a functional toxin-antitoxin system, but does not affect cell growth and might not affect the tolerance to isoniazid and rifampicin in M.smegmatis. Finally, the antitoxins of the known 5 TA systems are found not critical for auto-regulation of their promoter’s activity in M.smegmatis. These findings provide clues for further investigation of the roles of TA systems in M.tuberculosis.

Key words: Mycobacterium smegmatis, Toxoids, Antitoxins, Gene expression regulation, bacterial, Drug tolerance

ZHANG Lan-yue,GENG Yi-man,JIA Hong-yan,XIAO Jing,LI Zi-hui,PAN Li-ping,SUN Yi-cheng,ZHANG Zong-de. Preliminary study on the gene function of a novel toxin-antitoxin system MSMEG_3435-3436 in Mycobacterium smegmatis[J]. Chinese Journal of Antituberculosis, 2020, 42(2): 133-142. doi: 10.3969/j.issn.1000-6621.2020.02.010

Figures/Tables 9

材料类型	相关特点	来源
菌株
E.coli DH5α	克隆菌株	北京全式金生物技术有限公司
M.smegmatis	MC²155 野生株	本实验室保存
SY3807	含pJV53-Cpf1质粒的耻垢分枝杆菌	本研究
SY6248	敲除了MSMEG_3435-3436的耻垢分枝杆菌菌株	本研究
SY6249	敲除了MSMEG_6762-6760的耻垢分枝杆菌菌株	本研究
SY3126	含pJV53-GFP质粒的的耻垢分枝杆菌菌株	本研究
SY3328	MSMEG_1277-1278位置替换为LacZ的耻垢分枝杆菌菌株	本研究
SY3309	MSMEG_1283-1284位置替换为LacZ的耻垢分枝杆菌菌株	本研究
SY6407	MSMEG_3435-3436位置替换为LacZ的耻垢分枝杆菌菌株	本研究
SY3310	MSMEG_4447-4448位置替换为LacZ的耻垢分枝杆菌菌株	本研究
SY3311	MSMEG_5635-5634位置替换为LacZ的耻垢分枝杆菌菌株	本研究
质粒
pJV53	表达gp60 和gp61 重组蛋白的质粒,含卡那霉素抗性元件	文献[12]
pMV261	含Hsp60启动子的整合质粒,含卡那霉素抗性元件	文献[13]
pJV53-gfp	表达gp60 和gp61 重组蛋白的带有gfp基因,含卡那霉素抗性元件的质粒	文献[14]
pJV53-Cas12a	表达gp60 和gp61 重组蛋白的诱导Pmyc1tetO启动子控制 Cas12a 表达,含卡那霉素抗性元件的质粒	文献[14]
pCR-Hyg	crRNA插入Hsp60 启动子下游BpmⅠ和HindⅢ位点之间,含潮霉素抗性元件的质粒	文献[14]
pYC601	无水四环素诱导表达,含潮霉素抗性元件的质粒	文献[14]
pYC2034	MSMEG_3435-3436 crRNA插入pCR-Hyg的BpmⅠ和HindⅢ位点之间,含潮霉素抗性元件的质粒	本研究
pYC2033	MSMEG_6762-6760 crRNA插入pCR-Hyg的 BpmⅠ和HindⅢ位点之间,含潮霉素抗性元件的质粒	本研究
pYC2037	pUC19包含dsDNA同源臂以删除MSMEG_3435-3436,含氨苄青霉素抗性元件的质粒	本研究
pYC2036	pUC19包含dsDNA同源臂以删除MSMEG_6762-6760,含氨苄青霉素抗性元件的质粒	本研究
pYC2073	把MSMEG_3436基因放在pYC601的Pmyc1tetO启动子下,含潮霉素抗性元件的质粒	本研究
pYC2074	把MSMEG_3435-3436基因放在pYC601的Pmyc1tetO启动子下,含潮霉素抗性元件的质粒	本研究
pYC2076	把MSMEG_6760基因放在pYC601的Pmyc1tetO启动子下,含潮霉素抗性元件的质粒	本研究
pYC2077	把MSMEG_6762-6760基因放在pYC601的Pmyc1tetO启动子下,含潮霉素抗性元件的质粒	本研究

引物名称	引物序列(5'~3')
MSMEG_3436F	TCCGCATGCGGAGGAATCAGGTGAAACGACTCGATCTCGT
MSMEG_3436R	GTCCCCAATTAATTAGCTAACTATTCGGGCCACCGCGAGG
MSMEG_3436-3435F	TCCGCATGCGGAGGAATCAGATGACTGAGACCGCAGATCG
MSMEG_3436-3435R	GTCCCCAATTAATTAGCTAACTATTCGGGCCACCGCGAGG
MSMEG_6760F	TCCGCATGCGGAGGAATCAGATGCCCGTGACCGATGTGAA
MSMEG_6760R	GTCCCCAATTAATTAGCTAATCACGCAGTGAGCAGCGCGT
MSMEG_6760-6762F	TCCGCATGCGGAGGAATCAGGTGTGGGTGACCGTGATCGA
MSMEG_6760-6762R	GTCCCCAATTAATTAGCTAATCACGCAGTGAGCAGCGCGT
MSMEG_3435-3436 crRNA Top	ATGTCGAACTCACATTGGCCCCGCTGCA
MSMEG_3435-3436 crRNA Bottom	AGCTTGCAGCGGGGCCAATGTGAGTTCGACATCT
MSMEG_6760-6762 crRNA Top	ATACATCGGTCACGGGCATGTCAGTCCA
MSMEG_6760-6762 crRNA Bottom	AGCTTGGACTGACATGCCCGTGACCGATGTATCT
MSMEG_1277F	ATGCGGAGGAATCAGGATCCATGCCGTCGCTGAACATCGA
MSMEG_1277R	GTCCCCAATTAATTAGCTAATCACGCCAAGCGGCGGTTGA
MSMEG_1283F	ATGCGGAGGAATCAGGATCCATGGCTCTAAGCATCAAACA
MSMEG_1283R	GTCCCCAATTAATTAGCTAATCAGGACGGCAGACCGCGGT
MSMEG_3435F	ATGCGGAGGAATCAGGATCCATGACTGAGACCGCAGATCG
MSMEG_3435R	GTCCCCAATTAATTAGCTAATCACCGCTGCGCAGGAGCCA
MSMEG_4447F	ATGCGGAGGAATCAGGATCCATGACTCCCGCGCGTGACCG
MSMEG_4447R	GTCCCCAATTAATTAGCTAATTAGGCGTCGTCCCAGTCGAC
MSMEG_5635F	ATGCGGAGGAATCAGGATCCATGGGATTCCTGGACAAGGC
MSMEG_5635R	GTCCCCAATTAATTAGCTAATTACTGCTGTGGTTCTTGAG
MSMEG_3435 up HR F	TCTTCGCTATTACGCCAGCTTCCAGTTCAACCGAGACTGC
MSMEG_3435up HR R	CGCAATTGTCTTGGCCATGACACGCCTCCATGTAGCAGT
MSMEG_3435 down HR F	GGACTAGTTCGACGTTCTATGACAACAGCG
MSMEG_3435 down HR R	CCCAAGCTTATTTCGGTTTGGTGGTCCACT
LacZ F	ACTGCTACATGGAGGCGTGTCATGGCCAAGACAATTGCG
LacZ R	CCCGGGATCCGATATCTAGACTTATTTTTGACACCAGAC
UD-LacZ F	TCTAGATATCGGATCCCGGG
UD-LacZ R	AGCTGGCGTAATAGCGAAGA
F in Us for MSMEG_3435-3436+	AGTGAATTCGAGCTCGGTACCACCGGTGATCAACGTCAAC
R in Us for MSMEG_3435-3436	GAAGCGAGTGACCCGATCTG
F in Ds for MSMEG_3435-3436+	CAGATCGGGTCACTCGCTTCGAATAGATTGTCGCGGTTCAG
R in Ds for MSMEG_3435-3436+	TGCATGCCTGCAGGTCGACTGCCATTTCGCTTCGGTGAG
F in Us for MSMEG_6760-6762+	AGTGAATTCGAGCTCGGTACTGACCGACGAGTTGCTCGAAA
R in Us for MSMEG_6760-6762	TCGGCCATCAACCAGATCGA
F in Ds for MSMEG_6760-6762+	TCGATCTGGTTGATGGCCGACACACAACGTACAACCATCTG
R in Ds for MSMEG_6760-6762+	TGCATGCCTGCAGGTCGACTGATGTTCGGCACCAGGTTCT
MSMEG_3435 up F	CGTCCAGTTCAACCGAGACT
MSMEG_ 6760 up F	TACCGCGTGATGGCAAAGGA
F in Us for MSMEG_3435-3436	CACCGGTGATCAACGTCAAC
R in Ds for MSMEG_3435-3436	GCCATTTCGCTTCGGTGAG
F in Us for MSMEG_6760-6762	TGACCGACGAGTTGCTCGAAA
R in Ds for MSMEG_6760-6762	GATGTTCGGCACCAGGTTCT
F in down HR	GGACTAGTTCGACGTTCTATGACAACAGCG
R in down HR	CCCAAGCTTATTTCGGTTTGGTGGTCCACT
F	TCTAGATATCGGATCCCGGG
R	AGCTGGCGTAATAGCGAAGA
F in up HR	TCTTCGCTATTACGCCAGCTTCCAGTTCAACCGAGACTGC
R in up HR	CGCAATTGTCTTGGCCATGACACGCCTCCATGTAGCAGT

菌株类型	菌落形成单位(CFU, $x ˉ$ ±s)			处理后存活率(%, $x ˉ$ ±s)
菌株类型	处理前 (×10⁵)	异烟肼处理后 (×10⁵)	利福平处理后 (×10⁴)	异烟肼处理后	利福平处理后
野生株	126.70±13.33	5.33±1.76	1.80±0.46	4.38±1.48	0.15±0.04
突变株	266.70±6.67	9.33±1.76	0.33±0.13	3.49±0.66	0.03±0.02
t值	9.391	1.604	3.051	0.548	2.663
P值	0.001	0.184	0.038	0.613	0.056

菌株	吸光度值			时间 (min)	β-半乳糖苷酶活性(MU, $x ˉ$ ±s)	t值	P值
菌株	A₆₀₀值( $x ˉ$ ±s)	A₄₂₀值( $x ˉ$ ±s)	A₅₅₀值	时间 (min)	β-半乳糖苷酶活性(MU, $x ˉ$ ±s)	t值	P值
SY3328:: con	1.025±0.006	0.662±0.029	0.048	5.0	376.50±17.13	2.272	0.086
SY3328:: MSMEG_1277	0.991±0.027	0.550±0.025	0.047	5.0	315.50±20.71
SY3309:: con	1.018±0.016	0.318±0.015	0.050	4.0	189.00±12.24	1.795	0.147
SY3309:: MSMEG_1283	1.067±0.014	0.297±0.007	0.052	4.0	160.70±9.89
SY6407:: con	0.819±0.006	0.480±0.021	0.047	18.0	225.20±9.95	1.319	0.258
SY6407:: MSMEG_3435	0.815±0.003	0.476±0.006	0.047	19.0	211.70±2.57
SY3310: con	0.932±0.031	0.417±0.022	0.047	5.5	221.40±12.07	1.949	0.123
SY3310: MSMEG_4447	0.839±0.029	0.317±0.018	0.047	5.0	186.60±13.17
SY3311:: con	0.988±0.001	0.402±0.013	0.048	6.0	179.10±5.87	2.562	0.063
SY3311:: MSMEG_5635	0.944±0.037	0.290±0.030	0.052	5.5	127.70±19.21

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