Hanna Engelberg-kulka - Academia.edu (original) (raw)

Papers by Hanna Engelberg-kulka

mBio, 2016

Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requir... more Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) e xtracellular d eath f actor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans -translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans -translation is described as a regulatory system that attenuated the generation...

PLoS genetics, 2006

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms.... more Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin-antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.

Journal of bacteriology, 2004

mazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin. I... more mazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin. Inhibiting mazEF expression appeared to activate the module to cause cell death. Here we show that several stressful conditions, including high temperatures, DNA damage, and oxidative stress, also induce mazEF-mediated cell death. We also show that this process takes place only during logarithmic growth and requires an intact relA gene.

Communicative & integrative biology, 2009

The modes of action of antibiotics are mainly characterized by their effects on their targets. Pr... more The modes of action of antibiotics are mainly characterized by their effects on their targets. Previously,1,2 and in a recent paper,3 we have reported our discovery of a new mechanism for the action of some antibiotics. Rather than directly interfering with a vital bacterial pathway, these antibiotics act by triggering the bacterial toxin-antitoxin chromosomal module mazEF, thereby causing the bacteria to commit suicide. We also showed that antibiotics that inhibit transcription and/or translation cause mazEF-mediated cell death by forming Reactive Oxygen Species (ROS).3 Moreover, we found that after treatment by such antibiotics, the mazEF system cannot be activated, and thus ROS cannot be formed, without the presence of communication signaling peptide called the Extracellular Death Factor (EDF). Our results challenge the classical division between bacteriostatic and bactericidal antibiotics. Our study further provides evidence that mode of action of antibiotics may also be determi...

Nucleic acids research, 2015

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environme... more Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-t...

mBio, 2014

In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting i... more In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the F...

Trends in biochemical sciences, 2012

The bacterial stress response, a strategy to cope with environmental changes, is generally known ... more The bacterial stress response, a strategy to cope with environmental changes, is generally known to operate on the transcriptional level. Here, we discuss a novel paradigm for stress adaptation at the post-transcriptional level, based on the recent discovery of a stress-induced modified form of the translation machinery in Escherichia coli that is generated by MazF, the toxin component of the toxin-antitoxin (TA) module mazEF. Under stress, the induced endoribonuclease MazF removes the 3'-terminal 43 nucleotides of the 16S rRNA of ribosomes and, concomitantly, the 5'-untranslated regions (UTRs) of specific transcripts. This elegant mechanism enables selective translation due to the complementary effect of MazF on ribosomes and mRNAs, and also represents the first example of functional ribosome heterogeneity based on rRNA alteration.

Science (New York, N.Y.), Jan 25, 2003

The Journal of biological chemistry, Jan 25, 2003

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to cry... more A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.

Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates ce... more Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates cell death requiring the quorum-sensing pentapeptide NNWNN designated EDF (extracellular death factor). E. coli toxin MazF is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA sequences. E. coli ChpBK, a toxin homologous to MazF, is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA, ACG, and ACU sequences. Here we report that, in vitro, the signaling molecule EDF significantly amplifies the endoribonucleolytic activities of both MazF and ChpBK. EDF also overcomes the inhibitory activity of the antitoxins MazE over the toxin MazF and ChpBI over ChpBK. EDF sequence is important for both functions. Moreover, direct sequencespecific binding of EDF to MazF has been confirmed. Peptide-protein modeling revealed parallel contacts between EDF-MazF and MazE-MazF. These findings are intriguing, since most known quorum-sensing molecules monitor gene expression on the transcriptional level, while EDF monitors posttranscriptionally.

Molecular microbiology, Jan 13, 2015

The life cycle of phage λ has been studied extensively. Of particular interest has been the proce... more The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the E. coli toxin-antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of...

PloS one, 2014

The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized ... more The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage. It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage is inhibited by various elements involved in the expression of the E. coli toxin-antitoxin mazEF pathway. Since the mazEF module is present on the chromosomes of most E. coli strains, here we asked: Why is the SOS response found in so many E. coli strains? Is the mazEF module present but inactive in those strains? We examined three E. coli strains used for studies of the SOS response, strains AB1932, BW25113, and MG1655. We found that each of these strains is either missing or inhibiting one of several elements involved in the expression of the mazEF-mediated death pathway. Thus, the SOS response only takes place in E. coli cells in which one or more elements of the E. coli toxin-antitoxin module mazEF or its dow...

Trends in Biochemical Sciences - TRENDS BIOCHEM SCI, 1993

Trends in Microbiology, 2004

MGG Molecular & General Genetics, 1979

A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for s... more A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for suppression of a nonsense codon was studied for its ability to propagate phages T2, T4D, T5, ~K, f2, MS2, R17, Q/~, 2 as well as filamentous phages fl, fd and M13. Of all phages tested, only the growth of Qfl, 2, and filamentous phages is inhibited in the mutant at 42 ° C. This selective inhibition suggests that, like Qfi, 2 and filamentous phages also require a read-through protein(s) which results from suppression of a termination codon.

MGG Molecular & General Genetics, 1982

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in ... more We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

MGG Molecular & General Genetics, 1982

Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia... more Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia coli mutant LD1, we confirmed that LD1 is temperature-sensitive for suppression of nonsense codons, and that this phenotype of the mutant and its streptomycin-resistance are genetically linked and are probably caused by a single mutation, strA(LD1). We also isolated a spontaneous revertant, called LD1-R, which partially relieves the restriction of nonsense suppression caused by the strA(LD1) mutation. LD1-R is derived by an additional mutation (revA) which is closely linked to strA(LD1). We further demonstrate that the weak suppression of a lacZUGA mutation in a suppressor-free strain, which probably takes place by normal tRNA1rp, can be detected by the use of the chromagenic substance x-gal (5-Bromo-4-chloro-3-indolyl-beta-D-Galactopyranoside).

Science (New York, N.Y.), 2007

mazEF is a toxin-antitoxin module located on many bacterial chromosomes, including those of patho... more mazEF is a toxin-antitoxin module located on many bacterial chromosomes, including those of pathogens. Here, we report that Escherichia coli mazEF-mediated cell death is a population phenomenon requiring a quorum-sensing molecule that we call the extracellular death factor (EDF). Structural analysis revealed that EDF is a linear pentapeptide, Asn-Asn-Trp-Asn-Asn. Each of the five amino acids of EDF is important for its activity.

Cell, 1993

The E. coli trpR gene encodes the 108 amino acid long trp repressor. We have previously shown tha... more The E. coli trpR gene encodes the 108 amino acid long trp repressor. We have previously shown that a +1 frameshifting event occurs during the expression of trpR. Here we show that the transition from the 0 to the +1 frame of trpR occurs by the bypassing of a 55 nt long segment of the trpR+1-lacZ mRNA. This bypassing event is not pretranslational, and it probably takes place during translation. Two adjacent elements are required: a specific sequence of trpR, which must be preceded by a nonspecific 5' end longer than 10 translatable codons. Unique to trpR-lacZ bypassing is that the 55 nt long region must be translated in frame 0 to enable bypassing into the +1 frame. Translational bypassing as a newly discovered mechanism of gene expression is discussed, and the possible existence of translational introns is suggested.

mBio, 2016

Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requir... more Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) e xtracellular d eath f actor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans -translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans -translation is described as a regulatory system that attenuated the generation...

PLoS genetics, 2006

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms.... more Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin-antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.

Journal of bacteriology, 2004

mazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin. I... more mazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin. Inhibiting mazEF expression appeared to activate the module to cause cell death. Here we show that several stressful conditions, including high temperatures, DNA damage, and oxidative stress, also induce mazEF-mediated cell death. We also show that this process takes place only during logarithmic growth and requires an intact relA gene.

Communicative & integrative biology, 2009

The modes of action of antibiotics are mainly characterized by their effects on their targets. Pr... more The modes of action of antibiotics are mainly characterized by their effects on their targets. Previously,1,2 and in a recent paper,3 we have reported our discovery of a new mechanism for the action of some antibiotics. Rather than directly interfering with a vital bacterial pathway, these antibiotics act by triggering the bacterial toxin-antitoxin chromosomal module mazEF, thereby causing the bacteria to commit suicide. We also showed that antibiotics that inhibit transcription and/or translation cause mazEF-mediated cell death by forming Reactive Oxygen Species (ROS).3 Moreover, we found that after treatment by such antibiotics, the mazEF system cannot be activated, and thus ROS cannot be formed, without the presence of communication signaling peptide called the Extracellular Death Factor (EDF). Our results challenge the classical division between bacteriostatic and bactericidal antibiotics. Our study further provides evidence that mode of action of antibiotics may also be determi...

Nucleic acids research, 2015

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environme... more Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-t...

mBio, 2014

In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting i... more In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the F...

Trends in biochemical sciences, 2012

The bacterial stress response, a strategy to cope with environmental changes, is generally known ... more The bacterial stress response, a strategy to cope with environmental changes, is generally known to operate on the transcriptional level. Here, we discuss a novel paradigm for stress adaptation at the post-transcriptional level, based on the recent discovery of a stress-induced modified form of the translation machinery in Escherichia coli that is generated by MazF, the toxin component of the toxin-antitoxin (TA) module mazEF. Under stress, the induced endoribonuclease MazF removes the 3'-terminal 43 nucleotides of the 16S rRNA of ribosomes and, concomitantly, the 5'-untranslated regions (UTRs) of specific transcripts. This elegant mechanism enables selective translation due to the complementary effect of MazF on ribosomes and mRNAs, and also represents the first example of functional ribosome heterogeneity based on rRNA alteration.

Science (New York, N.Y.), Jan 25, 2003

The Journal of biological chemistry, Jan 25, 2003

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to cry... more A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.

Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates ce... more Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates cell death requiring the quorum-sensing pentapeptide NNWNN designated EDF (extracellular death factor). E. coli toxin MazF is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA sequences. E. coli ChpBK, a toxin homologous to MazF, is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA, ACG, and ACU sequences. Here we report that, in vitro, the signaling molecule EDF significantly amplifies the endoribonucleolytic activities of both MazF and ChpBK. EDF also overcomes the inhibitory activity of the antitoxins MazE over the toxin MazF and ChpBI over ChpBK. EDF sequence is important for both functions. Moreover, direct sequencespecific binding of EDF to MazF has been confirmed. Peptide-protein modeling revealed parallel contacts between EDF-MazF and MazE-MazF. These findings are intriguing, since most known quorum-sensing molecules monitor gene expression on the transcriptional level, while EDF monitors posttranscriptionally.

Molecular microbiology, Jan 13, 2015

The life cycle of phage λ has been studied extensively. Of particular interest has been the proce... more The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the E. coli toxin-antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of...

PloS one, 2014

The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized ... more The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage. It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage is inhibited by various elements involved in the expression of the E. coli toxin-antitoxin mazEF pathway. Since the mazEF module is present on the chromosomes of most E. coli strains, here we asked: Why is the SOS response found in so many E. coli strains? Is the mazEF module present but inactive in those strains? We examined three E. coli strains used for studies of the SOS response, strains AB1932, BW25113, and MG1655. We found that each of these strains is either missing or inhibiting one of several elements involved in the expression of the mazEF-mediated death pathway. Thus, the SOS response only takes place in E. coli cells in which one or more elements of the E. coli toxin-antitoxin module mazEF or its dow...

Trends in Biochemical Sciences - TRENDS BIOCHEM SCI, 1993

Trends in Microbiology, 2004

MGG Molecular & General Genetics, 1979

A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for s... more A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for suppression of a nonsense codon was studied for its ability to propagate phages T2, T4D, T5, ~K, f2, MS2, R17, Q/~, 2 as well as filamentous phages fl, fd and M13. Of all phages tested, only the growth of Qfl, 2, and filamentous phages is inhibited in the mutant at 42 ° C. This selective inhibition suggests that, like Qfi, 2 and filamentous phages also require a read-through protein(s) which results from suppression of a termination codon.

MGG Molecular & General Genetics, 1982

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in ... more We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

MGG Molecular & General Genetics, 1982

Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia... more Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia coli mutant LD1, we confirmed that LD1 is temperature-sensitive for suppression of nonsense codons, and that this phenotype of the mutant and its streptomycin-resistance are genetically linked and are probably caused by a single mutation, strA(LD1). We also isolated a spontaneous revertant, called LD1-R, which partially relieves the restriction of nonsense suppression caused by the strA(LD1) mutation. LD1-R is derived by an additional mutation (revA) which is closely linked to strA(LD1). We further demonstrate that the weak suppression of a lacZUGA mutation in a suppressor-free strain, which probably takes place by normal tRNA1rp, can be detected by the use of the chromagenic substance x-gal (5-Bromo-4-chloro-3-indolyl-beta-D-Galactopyranoside).

Science (New York, N.Y.), 2007

mazEF is a toxin-antitoxin module located on many bacterial chromosomes, including those of patho... more mazEF is a toxin-antitoxin module located on many bacterial chromosomes, including those of pathogens. Here, we report that Escherichia coli mazEF-mediated cell death is a population phenomenon requiring a quorum-sensing molecule that we call the extracellular death factor (EDF). Structural analysis revealed that EDF is a linear pentapeptide, Asn-Asn-Trp-Asn-Asn. Each of the five amino acids of EDF is important for its activity.

Cell, 1993

The E. coli trpR gene encodes the 108 amino acid long trp repressor. We have previously shown tha... more The E. coli trpR gene encodes the 108 amino acid long trp repressor. We have previously shown that a +1 frameshifting event occurs during the expression of trpR. Here we show that the transition from the 0 to the +1 frame of trpR occurs by the bypassing of a 55 nt long segment of the trpR+1-lacZ mRNA. This bypassing event is not pretranslational, and it probably takes place during translation. Two adjacent elements are required: a specific sequence of trpR, which must be preceded by a nonspecific 5' end longer than 10 translatable codons. Unique to trpR-lacZ bypassing is that the 55 nt long region must be translated in frame 0 to enable bypassing into the +1 frame. Translational bypassing as a newly discovered mechanism of gene expression is discussed, and the possible existence of translational introns is suggested.