假单胞菌Tw224抗砷基因簇的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0418

摘要/Abstract

摘要：

长期在砷污染环境生存的细菌逐渐演化出抗砷机制,旨在评价分离自湖南铅锌矿区污染土壤的Pseudomonas sp. Tw224抗砷能力和抗重金属谱;揭示该菌的抗砷基因/基因簇的功能。在砷或重金属存在条件下测量细菌的生长情况,对该菌的抗砷能力和抗重金属谱进行评价;通过基因组框架图分析该菌的抗砷基因/基因簇;采用代谢工程法构建基因簇缺失突变株揭示基因簇的功能。结果显示,砷延长了该菌的延迟期,降低了该菌的最高菌浓,该菌能够抗As⁵⁺和As³⁺的最高浓度分别为600 mmol/L和20 mmol/L,并且对铜、镍、锌、镉、铬、银和汞均具有不同程度的抗性。该菌基因组中共包含了20个与抗砷相关的基因（1个arsH,4个arsC,arsB和ACR（3）各2个,11个arsR）,其中arsH-arsC2-arsB-arsR（ars1）和arsR-arsB-arsC2（ars2）分别以基因簇的形式存在,长度分别为2.8 kb和2.2 kb。采用同源重组双交换法成功构建了ars1、ars2单缺失和ars1-ars2双缺失突变株Pseudomonas sp. QSA1、Pseudomonas sp. QSA2和Pseudomonas sp. QSARS。在无砷条件下3株突变株的生长均未受到影响,砷对QSA1和QSA2的最低抑菌浓度分别降低为野生菌Tw224的4%和80%;反应48 h,这两株突变株菌的砷还原能力分别约为野生菌的9.8%和53.8%。双基因簇突变株QSARS几乎完全失去了抗砷和砷的还原能力。Tw224具有很强的抗砷能力和广谱的抗重金属能力;基因组中存在大量抗重金属相关基因,其中有两个抗砷相关基因簇,ars1起主要的抗砷作用,ars2起辅助功能。

关键词: 假单胞菌, 基因簇, 砷耐受性, 基因敲除

Abstract:

The mechanisms of arsenic resistance have been gradually evolved in bacteria living in arsenic-contaminated environment. The purpose of this paper is to evaluate the resistance of Pseudomonas sp. Tw224 isolated from contaminated soil of lead-zinc mine in Hunan province to arsenic and its heavy metal-resistant spectrum,and to reveal its function of arsenic-resistant gene/gene clusters. The growth of the bacteria was measured in the presence of arsenic or heavy metals,and their resistances to arsenic and heavy metal-resistant spectrum were evaluated. The arsenic-resistant gene/gene clusters of the bacteria were analyzed by a draft sequence of genome. And finally the gene cluster-deleted mutants were constructed by metabolic engineering method to reveal the functions of the gene clusters. The delayed period prolonged,and the maximum bacterial concentration reduced in the presence of arsenic. The maximum As⁵⁺-resistant and As³⁺-resistant concentrations of Tw224 were 600 and 20 mmol/L,respectively. Moreover,Tw224 was also resistant to Cu,Ni,Zn,Cd,Cr,Ag and Hg at varying level. A total of 20 genes（1 arsH,4 arsC,2 arsB,2 ACR（3）and 11 arsR）related to arsenic resistance were found in the genome of the strain,and arsH-arsC2-arsB-arsR（ars1）and arsH-arsB-arsC2（ars2）existed in the form of gene clusters with the length of 2.8 kb and 2.2 kb,respectively. The ars1-inactivated Pseudomonas sp. QSA1,ars2-inactivated Pseudomonas sp. QSA2,and ars1-ars2-inactivated Pseudomonas sp. QSARS were successfully constructed by homologous recombinant double exchange method. In the absence of arsenic,the growths of the three mutants were not affected,and the minimum inhibitory concentrations of arsenic to QSA1 and QSA2 reduced to 4% and 80% of that of wild one,respectively. The arsenic reduction capacity of the two mutants was about 9.8% and 53.8% of that of wild one at 48 h,respectively. Pseudomonas sp. QSARS,a double gene clusters-deleted mutant,almost completely lost its resistance to arsenic and arsenic reduction ability. In conclusion,Tw224 had strong resistance to arsenic and a broad spectrum of resistance to heavy metals. There are a large number of heavy metal-resistant related genes in its genome,among which there are two arsenic-resistant related gene clusters. The ars1 gene cluster played a major role and the ars2 played an auxiliary role in arsenic-resistant process.

Key words: Pseudomonas, gene cluster, resistance to arsenic, gene knockout

孙靖雅, 马玉超. 假单胞菌Tw224抗砷基因簇的功能研究[J]. 生物技术通报, 2022, 38(1): 141-149.

SUN Jing-ya, MA Yu-chao. Functions of Arsenic-resistant Gene Cluster in Pseudomonas sp. Tw224[J]. Biotechnology Bulletin, 2022, 38(1): 141-149.

图/表 8

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质粒和菌株Plasmids and strains		特性Characteristic	来源Source
质粒 Plasmid	pK18mobsacB	Suicide plasmid vector,KanR	Stored in lab
	pSET152	Integrativeplasmid vector,Apr	Stored in lab
	pA18mobsacB	Apr	This study
	pA18QCA1	pA18 with a1 up/a1 down,Apr	This study
	pA18QCA2	pA18 with a2 up/a2 down,Apr	This study
菌株 Strain	Pseudomonas sp. Tw224	KanR、AmpR	From Hunan,stored in lab
	E. coli S17-1	KanR	This study
	E. coli S17-1-QCA1	E. coli S17-1 with pA18QCA1	This study
	E. coli S17-1-QCA2	E. coli S17-1 with pA18QCA2	This study
	Pseudomonas sp. QSA1	Tw224 ∆ars1	This study
	Pseudomonas sp. QSA2	Tw224 ∆ars2	This study
	Pseudomonas sp. QSARS	Tw224 ∆ars1 ars2	This study

质粒和菌株Plasmids and strains		特性Characteristic	来源Source
质粒 Plasmid	pK18mobsacB	Suicide plasmid vector,KanR	Stored in lab
	pSET152	Integrativeplasmid vector,Apr	Stored in lab
	pA18mobsacB	Apr	This study
	pA18QCA1	pA18 with a1 up/a1 down,Apr	This study
	pA18QCA2	pA18 with a2 up/a2 down,Apr	This study
菌株 Strain	Pseudomonas sp. Tw224	KanR、AmpR	From Hunan,stored in lab
	E. coli S17-1	KanR	This study
	E. coli S17-1-QCA1	E. coli S17-1 with pA18QCA1	This study
	E. coli S17-1-QCA2	E. coli S17-1 with pA18QCA2	This study
	Pseudomonas sp. QSA1	Tw224 ∆ars1	This study
	Pseudomonas sp. QSA2	Tw224 ∆ars2	This study
	Pseudomonas sp. QSARS	Tw224 ∆ars1 ars2	This study

引物名称 Primer	序列 Sequence（5'-3'）
Apr UP	cgcagctgtgctcgacgtGAAGATCCTTTGATCTTTTC
Apr DOWN	ttcggcaagcaggcatcgccatgATCAGCCGTCCAAATGC
MQa1U S	attcgagctcggtacccgggAGGACCAGGCTGAGGACA
MQa1U A	TATTTGGTAGACCGCTATTCG
MQa1D S	gaatagcggtctaccaaataCCCAAGCACTTGAAGAATGT
MQa1D A	taaaacgacggccagtgccaCTGCCGAGCAAAGCGTAT
NEW2U S	attcgagctcggtacccgggCAGGGACAAGGGAATGACG
NEW2U A	TTGACGACGGTCCACCAG
MQa2D S	ctggtggaccgtcgtcaaAGGCGCTGATTCTGTGGG
MQa2D A	taaaacgacggccagtgccaTCAATGAGGCTGCGGATG
YZ UP	CCCCAGGCTTTACACTTTATG
YZ DOWN	TGTGCTGCAAGGCGATTA
YZA1Y S	ACCGATGTGAGCGAAGCC
YZA1Y A	CCTCCTGGTCGTTACCCTGT
YZA2Y S	CAATCAGTTGTGGCGGTTTC
YZA2Y A	CATGCTCAGCTCCTTTCGA

引物名称 Primer	序列 Sequence（5'-3'）
Apr UP	cgcagctgtgctcgacgtGAAGATCCTTTGATCTTTTC
Apr DOWN	ttcggcaagcaggcatcgccatgATCAGCCGTCCAAATGC
MQa1U S	attcgagctcggtacccgggAGGACCAGGCTGAGGACA
MQa1U A	TATTTGGTAGACCGCTATTCG
MQa1D S	gaatagcggtctaccaaataCCCAAGCACTTGAAGAATGT
MQa1D A	taaaacgacggccagtgccaCTGCCGAGCAAAGCGTAT
NEW2U S	attcgagctcggtacccgggCAGGGACAAGGGAATGACG
NEW2U A	TTGACGACGGTCCACCAG
MQa2D S	ctggtggaccgtcgtcaaAGGCGCTGATTCTGTGGG
MQa2D A	taaaacgacggccagtgccaTCAATGAGGCTGCGGATG
YZ UP	CCCCAGGCTTTACACTTTATG
YZ DOWN	TGTGCTGCAAGGCGATTA
YZA1Y S	ACCGATGTGAGCGAAGCC
YZA1Y A	CCTCCTGGTCGTTACCCTGT
YZA2Y S	CAATCAGTTGTGGCGGTTTC
YZA2Y A	CATGCTCAGCTCCTTTCGA

菌株Strain	最低抑菌浓度MIC /（mmol·L^-1）
菌株Strain	As⁵⁺	As³⁺	Cr³⁺	Ag³⁺	Cd²⁺	Co²⁺	Ni²⁺	Hg²⁺	Zn²⁺	Cu²⁺
Tw224	600	20	5	3	2	3	12	3	12	20