is a Senior Investigator in the Cell Biology and Metabolism Branch of the National Institute of C... more is a Senior Investigator in the Cell Biology and Metabolism Branch of the National Institute of Child Health and Human Development. Since her discovery of the OxyS RNA, the identification and characterization of small RNAs in Escherichia coli has become an everincreasing focus of research by her group.
Proceedings of the National Academy of Sciences, 1999
The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular... more The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro , we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.
Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-c... more Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that t...
Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with... more Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.
The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxida... more The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxidant genes in response to hydrogen peroxide in Escherichia coli . Genetic and biochemical studies revealed that OxyR is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin 1 (Grx1). The gene encoding Grx1 is regulated by OxyR, thus providing a mechanism for autoregulation. The redox potential of OxyR was determined to be –185 millivolts, ensuring that OxyR is reduced in the absence of stress. These results represent an example of redox signaling through disulfide bond formation and reduction.
protein is heat stable and has been reported to exist as a pentamer (Kajitani et al., 1994) or a ... more protein is heat stable and has been reported to exist as a pentamer (Kajitani et al., 1994) or a hexamer (Franze de Fernandez et al., 1972; Kamen et al., 1972; Carmichael et
Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and tra... more Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.
Bacteria, yeast and cells of higher eukaryotes specifically induce the expression of genes encodi... more Bacteria, yeast and cells of higher eukaryotes specifically induce the expression of genes encoding antioxidant defenses when exposed to reactive oxygen species. Recent studies have also suggested that reactive oxygen intermediates play a role as second messengers in signal transduction pathways. Therefore, cells must possess regulators that sense oxidant signals and transduce the signals into changes in gene expression. This review provides an overview of the transcription factors in Escherichia coli, Saccharomyces cerevisiae and mammalian cells that govern the response to oxidative stress. Some of the regulators function primarily as regulators of antioxidant genes while other regulators of the oxidative stress response also regulate genes important for metal homeostasis or cell metabolism during aerobic or anaerobic growth.
The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory networ... more The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C-terminal output domain and is the first known response regulator involved in the con...
Escherichia coli strains were previously found to be susceptible to the antituberculosis drug iso... more Escherichia coli strains were previously found to be susceptible to the antituberculosis drug isonicotinic acid hydrazide (isoniazid [INH]) when they carried certain mutations that also sensitize them to peroxides: a deletion in oxyR, a redox-sensitive regulator of hydrogen peroxide-inducible genes, or mutations in both katG and ahpCF, OxyR-regulated genes encoding hydroperoxidase I, and an alkyl hydroperoxide reductase. To test whether INH, like peroxides, activates OxyR, the effect of INH on OxyR regulation was examined. Primer extension assays showed that transcription of the OxyR-regulated oxyS gene was not significantly induced by INH in wild-type cells, indicating that INH does not activate OxyR. However, the INH-susceptible katG ahpCF mutant strain was found to have constitutively high levels of oxyS transcription. This suggested that the lack of peroxidase expression in these strains allows endogenous oxidants to accumulate, and this leads not only to constitutive OxyR activation but also to INH susceptibility. Consistent with this concept, hydrogen peroxide or cumene hydroperoxide potentiated the INH susceptibilities of wild-type cells, while the antioxidant ascorbic acid protected the susceptible katG ahpCF mutant strain from INH. Superoxide radicals, generated by paraquat, did not enhance the INH susceptibilities of wild-type cells. Hydrogen peroxide also potentiated the INH susceptibilities of susceptible and resistant (katG mutant) Mycobacterium smegmatis strains. Our results suggest that INH is converted to a more active drug by reaction with peroxides and that the INH susceptibilities of enterobacteria and mycobacteria are mechanistically related.
The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function... more The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144(Delta), contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously c...
The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reduct... more The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonela typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-l and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.
OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and p... more OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and positively regulates the expression of proteins important for the defense against hydrogen peroxide in Eschericha coli and Salmonella typhimurium. Using random mutagenesis, we isolated ...
OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia ... more OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded σ s subunit of RNA polymerase. Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS. We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation.
▪ The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modif... more ▪ The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modification or stability, and of mRNA translation is becoming increasingly apparent. Here we discuss current knowledge of regulatory RNA function and review how the RNAs have been identified in a variety of organisms. Many of the regulatory RNAs act through base-pairing interactions with target RNAs. The base-pairing RNAs can be grouped into two general classes: those that are encoded on the opposite strand of their target RNAs such that they contain perfect complementarity with their targets, and those that are encoded at separate locations on the chromosome and have imperfect base-pairing potential with their targets. Other regulatory RNAs act by modifying protein activity, in some cases by mimicking the structures of other RNA or DNA molecules.
is a Senior Investigator in the Cell Biology and Metabolism Branch of the National Institute of C... more is a Senior Investigator in the Cell Biology and Metabolism Branch of the National Institute of Child Health and Human Development. Since her discovery of the OxyS RNA, the identification and characterization of small RNAs in Escherichia coli has become an everincreasing focus of research by her group.
Proceedings of the National Academy of Sciences, 1999
The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular... more The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro , we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.
Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-c... more Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that t...
Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with... more Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.
The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxida... more The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxidant genes in response to hydrogen peroxide in Escherichia coli . Genetic and biochemical studies revealed that OxyR is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin 1 (Grx1). The gene encoding Grx1 is regulated by OxyR, thus providing a mechanism for autoregulation. The redox potential of OxyR was determined to be –185 millivolts, ensuring that OxyR is reduced in the absence of stress. These results represent an example of redox signaling through disulfide bond formation and reduction.
protein is heat stable and has been reported to exist as a pentamer (Kajitani et al., 1994) or a ... more protein is heat stable and has been reported to exist as a pentamer (Kajitani et al., 1994) or a hexamer (Franze de Fernandez et al., 1972; Kamen et al., 1972; Carmichael et
Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and tra... more Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.
Bacteria, yeast and cells of higher eukaryotes specifically induce the expression of genes encodi... more Bacteria, yeast and cells of higher eukaryotes specifically induce the expression of genes encoding antioxidant defenses when exposed to reactive oxygen species. Recent studies have also suggested that reactive oxygen intermediates play a role as second messengers in signal transduction pathways. Therefore, cells must possess regulators that sense oxidant signals and transduce the signals into changes in gene expression. This review provides an overview of the transcription factors in Escherichia coli, Saccharomyces cerevisiae and mammalian cells that govern the response to oxidative stress. Some of the regulators function primarily as regulators of antioxidant genes while other regulators of the oxidative stress response also regulate genes important for metal homeostasis or cell metabolism during aerobic or anaerobic growth.
The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory networ... more The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C-terminal output domain and is the first known response regulator involved in the con...
Escherichia coli strains were previously found to be susceptible to the antituberculosis drug iso... more Escherichia coli strains were previously found to be susceptible to the antituberculosis drug isonicotinic acid hydrazide (isoniazid [INH]) when they carried certain mutations that also sensitize them to peroxides: a deletion in oxyR, a redox-sensitive regulator of hydrogen peroxide-inducible genes, or mutations in both katG and ahpCF, OxyR-regulated genes encoding hydroperoxidase I, and an alkyl hydroperoxide reductase. To test whether INH, like peroxides, activates OxyR, the effect of INH on OxyR regulation was examined. Primer extension assays showed that transcription of the OxyR-regulated oxyS gene was not significantly induced by INH in wild-type cells, indicating that INH does not activate OxyR. However, the INH-susceptible katG ahpCF mutant strain was found to have constitutively high levels of oxyS transcription. This suggested that the lack of peroxidase expression in these strains allows endogenous oxidants to accumulate, and this leads not only to constitutive OxyR activation but also to INH susceptibility. Consistent with this concept, hydrogen peroxide or cumene hydroperoxide potentiated the INH susceptibilities of wild-type cells, while the antioxidant ascorbic acid protected the susceptible katG ahpCF mutant strain from INH. Superoxide radicals, generated by paraquat, did not enhance the INH susceptibilities of wild-type cells. Hydrogen peroxide also potentiated the INH susceptibilities of susceptible and resistant (katG mutant) Mycobacterium smegmatis strains. Our results suggest that INH is converted to a more active drug by reaction with peroxides and that the INH susceptibilities of enterobacteria and mycobacteria are mechanistically related.
The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function... more The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144(Delta), contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously c...
The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reduct... more The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonela typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-l and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.
OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and p... more OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and positively regulates the expression of proteins important for the defense against hydrogen peroxide in Eschericha coli and Salmonella typhimurium. Using random mutagenesis, we isolated ...
OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia ... more OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded σ s subunit of RNA polymerase. Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS. We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation.
▪ The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modif... more ▪ The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modification or stability, and of mRNA translation is becoming increasingly apparent. Here we discuss current knowledge of regulatory RNA function and review how the RNAs have been identified in a variety of organisms. Many of the regulatory RNAs act through base-pairing interactions with target RNAs. The base-pairing RNAs can be grouped into two general classes: those that are encoded on the opposite strand of their target RNAs such that they contain perfect complementarity with their targets, and those that are encoded at separate locations on the chromosome and have imperfect base-pairing potential with their targets. Other regulatory RNAs act by modifying protein activity, in some cases by mimicking the structures of other RNA or DNA molecules.
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Papers by G. Storz