Functions of Arsenic-resistant Gene Cluster in Pseudomonas sp. Tw224

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

Abstract

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

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

Figures/Tables 8

References 24

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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