Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: Homologies between OxyR protein and a family of bacterial activator proteins

Tao, Kazuyuki; Makino, Kae; Yonei, Shuji; Nakata, Atsuo; Shinagawa, Hideo

doi:10.1007/bf00332397

Cited by 94 publications

(43 citation statements)

References 30 publications

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“…In this study we show that the redox-sensitive transcriptional activator OxyR, a member of the LysR-type family of bacterial transcriptional activators (9,36), is responsible for the control of the peroxide response regulon in this anaerobic microorganism. This is the first description of a functional oxidative stress response regulator in obligate anaerobic bacteria and in one so greatly diverged from the main line of eubacterial descent (41).…”

Section: Discussionmentioning

confidence: 81%

The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe Bacteroides fragilis

2000

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The peroxide response-inducible genes ahpCF, dps, and katB in the obligate anaerobe Bacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length. dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR abolished the induction of ahpC and katB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in ⌬oxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of ⌬oxyR mutant strains with wildtype oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of the ahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in ⌬oxyR and overexpressing oxyR strains by Northern blotting and oxyR::xylB fusions revealed that B. fragilis OxyR does not control its own expression.The human intestinal obligate anaerobe Bacteroides fragilis possesses a complex oxidative stress response mechanism which is required to maintain extended aerotolerance compared to control cultures (24). A set of approximately 28 proteins are synthesized in response to treatment with hydrogen peroxide or oxygen exposure, but other proteins are also down regulated following a shift to aerobic conditions, and their role in the physiological adaptation to this adverse environment still remains unclear (24). The catalase gene katB is typical of the B. fragilis oxidative stress genes and is induced in mid-log phase following the addition of hydrogen peroxide or exposure to molecular oxygen or after entering the stationary phase (25). A katB mutant was found to be more sensitive to exogenous hydrogen peroxide under anaerobic conditions than was the parent strain, but aerotolerance in the presence of atmospheric oxygen was not significantly altered (24). The studies on resistance to peroxides led to the isolation of a KatB-overproducing...

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Section: Discussionmentioning

confidence: 81%

The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe Bacteroides fragilis

2000

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“…A recent map of the E. coli chromosome (22) suggested that the oxyR gene was located near the arg operon at 89.4 min; moreover, oxyR has recently been cloned and sequenced from the A 4G11 phage (40). Since the deduced oxyR restriction map perfectly matches that of the pMC7 plasmid from BamHI (bp 6850) to EcoRV (bp 8320), it has been proposed to map close to the arg operon (3,45).…”

Section: Resultsmentioning

confidence: 99%

“…To confirm this localization of the oxyR gene, we performed the following hybridization analysis. An oligonucleotide, whose sequence was derived from the oxyR DNA sequence (3,6,40,45), was hybridized to either restricted pMC7 plasmid DNA or restricted chromosomal DNA. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural and biochemical characterization of the Escherichia coli argE gene product

et al. 1992

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The DNA sequence of a 2,100-bp region containing the argE gene from Escherichia coli has been determined.The nucleotide sequence of the ppc-argE intergenic region was also solved and shown to contain six tandemly repeated REP sequences. Moreover, the oxyR gene has been mapped on the E. coli chromosome and shown to flank the arg operon. The codon responsible for the translation start of argE was determined by using site-directed mutants. This gene spans 1,400 bp and encodes a 42,350-Da polypeptide. The argE3 allele and a widely used argE amber gene have also been cloned and sequenced. N-Acetylornithinase, the argE product, has been overproduced and purified to homogeneity. Its main biochemical and catalytic properties are described. Moreover, we demonstrate that the protein is composed of two identical subunits. Finally, the amino acid sequence of N-acetylornithinase is shown to display a high degree of identity with those of the succinyldiaminopimelate desuccinylase from E. coli and carboxypeptidase G2 from a Pseudomonas sp. It is proposed that this carboxypeptidase might be responsible for the acetylornithinase-related activity found in the Pseudomonas sp.

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“…amylovora RcsA protein was compared to the consensus sequence of the LysR family of bacterial activator proteins. The LysR family consists of at least 10 different bacterial proteins known to act as transcriptional activators (Henikoff et al, 1988;Tao et al, 1989), with each protein having a conserved helixturn-helix DNA binding motif near to its N-terminus. The observations that the Er.…”

Section: Discussionmentioning

confidence: 99%

“…Computer-aided searches (Lipman & Pearson, 1985) of the NBRF, SWISSPROT and NEW NBRF protein databases failed to identify significant homology to any other protein sequences. In particular, no homology was found to either the LysR family of bacterial activator proteins (Henikoff, et al, 1988 ;Tao, et al, 1989) or to Cro-like DNA-binding proteins (Dodd & Egan, 1987).…”

Section: Methodsmentioning

confidence: 98%

Molecular cloning, expression and nucleotide sequence of the resA gene of Erwinia amylovora, encoding a positive regulator of capsule expression: evidence for a family of related capsule activator proteins

Coleman

Pearce

Hitchin

et al. 1990

Journal of General Microbiology

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A gene encoding a positive activator of the expression of extracellular polysaccharide (EPS) synthesis in the phytopathogen Erwinia amylouora has been isolated from a genomic library in Escherichia coli. The presence of the cloned gene in E. culi stimulated transcription of the genes encoding colanic acid biosynthesis and could complement rcsA mutations. Introduction of the gene on a multicopy plasmid into Er. amylouora caused a threefold increase in EPS expression. The nucleotide sequence of the gene (designated VCSA) was determined. This revealed a single open reading frame encoding an RcsA protein of 23.7 kDa. This was confirmed by minicell analysis in E. coli.The predicted amino acid sequence of this RcsA protein showed a high degree of homology to the RcsA protein of Klebsiella aerogenes, demonstrating the existence of a family of related RcsA activator proteins capable of stimulating EPS expression. The protein had no significant homology to known DNA-binding activator proteins, indicating, for the first time, that the RcsA family of activator proteins may stimulate expression of EPS synthesis indirectly by acting on other regulatory proteins.

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Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: Homologies between OxyR protein and a family of bacterial activator proteins

Cited by 94 publications

References 30 publications

The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe Bacteroides fragilis

The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe Bacteroides fragilis

Structural and biochemical characterization of the Escherichia coli argE gene product

Molecular cloning, expression and nucleotide sequence of the resA gene of Erwinia amylovora, encoding a positive regulator of capsule expression: evidence for a family of related capsule activator proteins

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