• Journal of Microbiology
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• 제35권4호
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• pp.277-283
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• 1997

Promoters inducible by paraquat, a superocide-generating agent, were isolated from Escherichia coli using a promoter-probing plasmid pRS415 with promoterless lacA gene. Twenty one promoters induced by paraquat were selected and further characterized. From sequence analysis, thirteen of the promoters were mapped to their specific loci on the Escherichia coli chromosome. Several promoters were mapped to the upstream of known genes such as usgl, katG, and mglB, whose relationships with superoxide response have not been previously reported. Other promoters were mapped to the upstream region of unknown open reading frames. Downstream of HC 96 promoter are uncharacterized ORFs whose sequences are homologous to ABC-transporter subunits. Downstream of HC84 promoter is an ORF encoding a transcriptional regulator-like protein, which contains a LysR family-specific HTH (helix-turn-helix) DNA bindign motif. We investigated whether these promoters belong to the soxRS regulon. All promoters except HC96 were found to belong to the soxRS regulon. The HC96 promoter was significantly induced by paraquat in the soxRS deletion mutant strain. The basal transcription level of three promoters (HE43, HC71, HD94) significantly increased at the stationary phase, implying that they are regulated by RpoS. However, paraquat inducibility of all promoters disappeared in the stationary phase, suggesting that SoxRS regulatory system is active only in rapidly growing cells.

• 미생물학회지
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• 제31권4호
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• pp.267-273
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• 1993

Escherichia coli 의 superoxide 라디칼 유도발현성 유전자의 탐색을 위하여 먼저 superoxide 라디칼에 의하여 발현이 유도되는 프로모터를 탐색하였다. 이를 위햐여, 프로모터 탐색벡터인 pJAC4 를 이용하여 E. coli MG1655 균주로부터 프로모터 library 를 구성하였다. Ampicillin 농도구 배정판법을 이용하여 프로모터 세기가 다른 클론들을 구별하여 프로모터 세기가 약한 것부터 중간정도까지 383개의 클론을 선별하였다. 이들 프로모터 클론들의 superoxide 라디칼 유도발현성을 paraquat 를 첨가한 ampicilin 농도구배평판에서 조사하였다. 이중 3개의 클론이 paraquat 에 의하여 유도됨을 확인하였고, 유도배율은 1.4-4배 정도이었다. 이들 프로모터의 염기서열을 결정한 결과 기종의 database 에 등록되어 있는 E. coli 염기서열들과는 다른 새로운 유전자임을 알았다. 이들 프로모터에 대하여 primer 연장법과 SS1 뉴클리아제 분석을 총하여 전사개시 자리를 결정하였고, paraquat 처리시 mRNA 의 양이 증가함을 관찰하였다. 특히 클론5의 경우는 두개의 전사개시 위치를 가지고 있으며, paraquat 처리시 상위 부분의 전사개시자리가 성택적으로 사용됨을 알 수 있었다. 프로모터 서열을 검색한 결과, RNA 중합효소($E\sigma^{70}$)에 의햐여 인지되는 -10과 -35부위를 가진 클론은 클론 5 뿐이었고, 나머지는 보존된 염기서열을 찾을 수 없었다. 이는 이들 프로모터들이 독특한 부류에 속하는 종류들로서, 새로운 전사조절인자를 요구할 가능성을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.386-390
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• 2006

A green fluorescent protein-based Pseudomonas putida reporter was successfully constructed and shown to be capable of detecting oxidative stress. In this whole-cell reporter, the promoter of the paraquat-inducible ferredoxin-$NADP^+$ reductase (fpr) was fused to a promoterless gfp gene on a broad-host-range promoter probe vector. Pseudomonas putida KT2440 harboring this reporter plasmid exhibited an increased level of gfp expression in the presence of redox-cycling agents (paraquat and menadione), hydrogen peroxide, and potential environmental pollutant chemicals such as toluene, paint thinner, gasoline, and diesel. Induction of fpr in the presence of these chemicals was confirmed using Northern blot analysis.

• Journal of Microbiology
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• 제34권2호
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• pp.137-143
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• 1996

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

• Journal of Microbiology
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• 제41권2호
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• pp.109-114
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• 2003

A promoter that is inducible by paraquat and menadione, the superoxide generators, independently of soxRS has been found in front of the sufABCDSE operon in Escherichia coli. Based on the observation that SufA is a holomog of IscA that functions in the assembly of iron sulfur cluster and the sufA promoter (sufAp) contains a putative Fur-binding consensus, we investigated whether this gene is regulated by Fur, a ferric uptake regulator, When examined in several sufAp-lacZ chromosomal fusion strains, sufAp was induced by EDTA, an iron chelator and a well-known Fur-inducer, The basal level of sufA expression increased dramatically in fur mutant, suggesting repression of sufAp by Fur. The derepression in fur mutant and EDTA-induction of sufA expression required nucleotides up to -61, where a putative Fur box is located. Purified Fur protein bound to the DNA fragment containing the putative Fur box between -35 and -10 promoter elements. The regulation by Fur and menadione induction of sufAp acted independently. The rpoS mutation increased sufA induction by menadione, suggesting that the stationary sigma factor RpoS acts negatively on sufA induction.