Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including trans... more Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including transient changes in transcription, translation, and posttranslation activities or heritable changes in regulatory or coding DNA sequences. This article addresses mechanisms for high-frequency, stochastic genotypic switching that occurs at DNA regions of increased recombination or mutation, called contingency loci. The heritable DNA rearrangements or mutations that occur at contingency loci pre-adapt a subpopulation of cells for variation in their environment. The systems that are highlighted are the defining examples for control of alternation of gene expression by site-specific DNA inversion, reversible insertion of an IS element, and slipped-strand mispairing at simple sequence DNA repeats.
Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to spe... more Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to specific promoters. Two families of sigma factors determine promoter specificity, the 70 (RpoD) family and the 54 (RpoN) family. In transcription controlled by 54 σ σ σ , the E 54 σ-promoter closed complex requires ATP hydrolysis by an associated bacterial enhancer-binding protein (bEBP) for the transition to open complex and transcription initiation. Given the wide host range of Salmonella enterica serovar Typhimurium, it is an excellent model system for investigating the roles of RpoN and its bEBPs in modulating the lifestyle of bacteria. The genome of S. Typhimurium encodes 13 known or predicted bEBPs, each responding to a unique intracellular or extracellular signal. While the regulons of most alternative sigma factors respond to a specific environmental or developmental signal, the RpoN regulon is very diverse, controlling genes for response to nitrogen limitation, nitric oxide stress, availability of alternative carbon sources, phage shock/envelope stress, toxic levels of zinc, nucleic acid damage, and other stressors. This review explores how bEBPs respond to environmental changes encountered by S. Typhimurium during transmission/infection and influence adaptation through control of transcription of different components of the S. Typhimurium RpoN regulon.
Background: Interactions between transcription factors and their specific binding sites are a key... more Background: Interactions between transcription factors and their specific binding sites are a key component of regulation of gene expression. Until recently, it was generally assumed that most bacterial transcription factor binding sites are located at or near promoters. However, several recent works utilizing high-throughput technology to detect transcription factor binding sites in bacterial genomes found a large number of binding sites in unexpected locations, particularly inside genes, as opposed to known or expected promoter regions. While some of these intragenic binding sites likely have regulatory functions, an alternative scenario is that many of these binding sites arise by chance in the absence of selective constraints. The latter possibility was supported by in silico simulations for σ 54 binding sites in Salmonella. Results: In this work, we extend these simulations to more than forty transcription factors from E. coli and other bacteria. The results suggest that binding sites for all analyzed transcription factors are likely to arise throughout the genome by random genetic drift and many transcription factor binding sites found in genomes may not have specific regulatory functions. In addition, when comparing observed and expected patterns of occurrence of binding sites in genomes, we observed distinct differences among different transcription factors. Conclusions: We speculate that transcription factor binding sites randomly occurring throughout the genome could be beneficial in promoting emergence of new regulatory interactions and thus facilitating evolution of gene regulatory networks.
Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertio... more Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine-or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.
Proceedings of the National Academy of Sciences of the United States of America, Feb 6, 2007
DNA rearrangements, including insertions, deletions, and inversions, control gene expression in n... more DNA rearrangements, including insertions, deletions, and inversions, control gene expression in numerous prokaryotic and eukaryotic systems, ranging from phase variation of surface antigens in pathogenic bacteria to generation of Ig diversity in human B cells. We report here that precise excision of the mobile element IS492 from one site on the Pseudoalteromonas atlantica chromosome directly correlates with phase variation of peripheral extracellular polysaccharide (p EPS) production from OFF (epsG::IS492) to ON (epsG ؉). In a previously undescribed application of quantitative PCR, we determined that the frequency of this transposase-dependent precise excision is remarkably high, ranging from 10 ؊3 to 10 ؊2 per cell per generation. High-frequency excision resulting in nonmutagenic repair of donor DNA is extremely unusual for classical transposable elements. Interestingly, high-frequency precise excision of IS492 does not occur at four different insertion sites on the P. atlantica chromosome, despite identity in the IS492 nucleotide sequences and 5-to 7-bp flanking DNA. The genome sequence revealed that epsG-associated IS492 is the only element inserted within a gene. Quantitative RT-PCR assays for externally derived transposase transcripts from each IS492 copy showed that IS492 at epsG has higher levels of host-initiated transcription through the element, suggesting that transcription per se or an increase in transposase (mooV) expression is responsible for the effect of chromosomal position on element excision. MooV levels and excision activity for IS492 inserted in forward and reverse orientations relative to plac and pT7 in Escherichia coli support that external transcription of mooV boosts transposase to a critical level required for detectable excision. DEDD-motif recombinases ͉ IS110 family ͉ phase-variation frequency ͉ transposition ͉ quantitative PCR Results Precise Excision of IS492 from epsG in P. atlantica Occurs at an Unusually High Frequency. It has been reported that P. atlantica DB27 colony switching from crenated (p EPS Ϫ) to mucoid (p EPS ϩ) Author contributions: B.P.H.
Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significan... more Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ 54 regulon in a bacterium is that the σ 54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ 54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all σ 54-dependent promoters for the characterization of the σ 54 regulon of Salmonella Typhimurium LT2. Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ 54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ 54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ 54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ 54 binding sites suggest that many of these sites are capable of functioning as σ 54-dependent promoters. Conclusion: Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ 54dependent promoters of the S. Typhimurium LT2 σ 54 regulon under a single growth condition, this approach is likely to be valuable for examining σ 54 regulons in other bacterial species. The S. Typhimurium σ 54 regulon included a high number of intragenic σ 54 binding sites/promoters, suggesting that σ 54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome... more Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome controls peripheral extracellular polysaccharide production and biofilm formation by P. atlantica. High-frequency precise excision of IS492 from epsG requires 5 and 7 bp of flanking DNA, suggesting that IS492 transposition involves a site-specific recombination mechanism. The site specificity of IS492 insertion was examined in P. atlantica and shown to be specific for a 7-bp target, 5-CTTGTTA-3. Characterization of numerous insertion events at the target site in epsG indicated that insertion is also orientation specific. The frequency of IS492 insertion at the epsG target site (2.7 ؋ 10 ؊7 /cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS492 precise excision from the same site. Comparison of insertion sites for IS492 and the highly related ISPtu2 from Pseudoalteromonas tunicata suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS492.
The 54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon ... more The 54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3=-phosphate cyclase (RtcA). Transcription from 54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the 54-dependent promoter of the rsr-yrlBA-rtcBA operon is RtcR. To identify conditions that generate the signal for RtcR activation in S. Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcRdependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion of rtcR or rtcB resulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module, dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon of Escherichia coli (rtcBA) did not stimulate expression of the S. Typhimurium RNA repair operon. Similarly, MMC did not induce expression of the E. coli rtcBA operon, although when expressed in S. Typhimurium, E. coli RtcR responds effectively to the unknown signal(s) generated there by MMC exposure. IMPORTANCE Homologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the 54-dependent RNA repair operon (rsr-yrlBA-rtcBA) of S. Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon components in vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.
Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including trans... more Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including transient changes in transcription, translation, and posttranslation activities or heritable changes in regulatory or coding DNA sequences. This article addresses mechanisms for high-frequency, stochastic genotypic switching that occurs at DNA regions of increased recombination or mutation, called contingency loci. The heritable DNA rearrangements or mutations that occur at contingency loci pre-adapt a subpopulation of cells for variation in their environment. The systems that are highlighted are the defining examples for control of alternation of gene expression by site-specific DNA inversion, reversible insertion of an IS element, and slipped-strand mispairing at simple sequence DNA repeats.
The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, Rtc... more The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria . In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon.
A regulatory scheme was revealed in which AalR responds to l -Asp and DarR responds to d -Asp, a ... more A regulatory scheme was revealed in which AalR responds to l -Asp and DarR responds to d -Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi .
Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the tra... more Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertio... more Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was address...
Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of t... more Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene famil...
Figure S1. Complete set of 'report cards' for transcription factor binding sites investig... more Figure S1. Complete set of 'report cards' for transcription factor binding sites investigaed in this work. (PDF 3337 kb)
20 Organismal adaptations to environmental stimuli are governed by intracellular signaling molecu... more 20 Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as 21 nucleotide second messengers. Recent studies have identified functional roles for the non-canonical 2 ́,3 ́22 cyclic nucleotide monophosphates (2 ́,3 ́-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli, 23 2 ́,3 ́-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate 24 biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli 25 and Salmonella Typhimurium that are modulated by 2 ́,3 ́-cNMPs through the development of cell26 permeable 2 ́,3 ́-cNMP analogs and a 2 ́,3 ́-cyclic nucleotide phosphodiesterase. Utilization of these 27 chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified 28 pathways regulated by 2 ́,3 ́-cNMPs, including flagellar motility and biofilm formation, and by 29 oligoribonucleotides with 3’-terminal 2 ́,3...
Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significan... more Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ regulon in a bacterium is that the σ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ-dependent promoters for the characterization of the σ regulon of Salmonella Typhimurium LT2. Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcr...
Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including trans... more Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including transient changes in transcription, translation, and posttranslation activities or heritable changes in regulatory or coding DNA sequences. This article addresses mechanisms for high-frequency, stochastic genotypic switching that occurs at DNA regions of increased recombination or mutation, called contingency loci. The heritable DNA rearrangements or mutations that occur at contingency loci pre-adapt a subpopulation of cells for variation in their environment. The systems that are highlighted are the defining examples for control of alternation of gene expression by site-specific DNA inversion, reversible insertion of an IS element, and slipped-strand mispairing at simple sequence DNA repeats.
Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to spe... more Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to specific promoters. Two families of sigma factors determine promoter specificity, the 70 (RpoD) family and the 54 (RpoN) family. In transcription controlled by 54 σ σ σ , the E 54 σ-promoter closed complex requires ATP hydrolysis by an associated bacterial enhancer-binding protein (bEBP) for the transition to open complex and transcription initiation. Given the wide host range of Salmonella enterica serovar Typhimurium, it is an excellent model system for investigating the roles of RpoN and its bEBPs in modulating the lifestyle of bacteria. The genome of S. Typhimurium encodes 13 known or predicted bEBPs, each responding to a unique intracellular or extracellular signal. While the regulons of most alternative sigma factors respond to a specific environmental or developmental signal, the RpoN regulon is very diverse, controlling genes for response to nitrogen limitation, nitric oxide stress, availability of alternative carbon sources, phage shock/envelope stress, toxic levels of zinc, nucleic acid damage, and other stressors. This review explores how bEBPs respond to environmental changes encountered by S. Typhimurium during transmission/infection and influence adaptation through control of transcription of different components of the S. Typhimurium RpoN regulon.
Background: Interactions between transcription factors and their specific binding sites are a key... more Background: Interactions between transcription factors and their specific binding sites are a key component of regulation of gene expression. Until recently, it was generally assumed that most bacterial transcription factor binding sites are located at or near promoters. However, several recent works utilizing high-throughput technology to detect transcription factor binding sites in bacterial genomes found a large number of binding sites in unexpected locations, particularly inside genes, as opposed to known or expected promoter regions. While some of these intragenic binding sites likely have regulatory functions, an alternative scenario is that many of these binding sites arise by chance in the absence of selective constraints. The latter possibility was supported by in silico simulations for σ 54 binding sites in Salmonella. Results: In this work, we extend these simulations to more than forty transcription factors from E. coli and other bacteria. The results suggest that binding sites for all analyzed transcription factors are likely to arise throughout the genome by random genetic drift and many transcription factor binding sites found in genomes may not have specific regulatory functions. In addition, when comparing observed and expected patterns of occurrence of binding sites in genomes, we observed distinct differences among different transcription factors. Conclusions: We speculate that transcription factor binding sites randomly occurring throughout the genome could be beneficial in promoting emergence of new regulatory interactions and thus facilitating evolution of gene regulatory networks.
Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertio... more Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine-or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.
Proceedings of the National Academy of Sciences of the United States of America, Feb 6, 2007
DNA rearrangements, including insertions, deletions, and inversions, control gene expression in n... more DNA rearrangements, including insertions, deletions, and inversions, control gene expression in numerous prokaryotic and eukaryotic systems, ranging from phase variation of surface antigens in pathogenic bacteria to generation of Ig diversity in human B cells. We report here that precise excision of the mobile element IS492 from one site on the Pseudoalteromonas atlantica chromosome directly correlates with phase variation of peripheral extracellular polysaccharide (p EPS) production from OFF (epsG::IS492) to ON (epsG ؉). In a previously undescribed application of quantitative PCR, we determined that the frequency of this transposase-dependent precise excision is remarkably high, ranging from 10 ؊3 to 10 ؊2 per cell per generation. High-frequency excision resulting in nonmutagenic repair of donor DNA is extremely unusual for classical transposable elements. Interestingly, high-frequency precise excision of IS492 does not occur at four different insertion sites on the P. atlantica chromosome, despite identity in the IS492 nucleotide sequences and 5-to 7-bp flanking DNA. The genome sequence revealed that epsG-associated IS492 is the only element inserted within a gene. Quantitative RT-PCR assays for externally derived transposase transcripts from each IS492 copy showed that IS492 at epsG has higher levels of host-initiated transcription through the element, suggesting that transcription per se or an increase in transposase (mooV) expression is responsible for the effect of chromosomal position on element excision. MooV levels and excision activity for IS492 inserted in forward and reverse orientations relative to plac and pT7 in Escherichia coli support that external transcription of mooV boosts transposase to a critical level required for detectable excision. DEDD-motif recombinases ͉ IS110 family ͉ phase-variation frequency ͉ transposition ͉ quantitative PCR Results Precise Excision of IS492 from epsG in P. atlantica Occurs at an Unusually High Frequency. It has been reported that P. atlantica DB27 colony switching from crenated (p EPS Ϫ) to mucoid (p EPS ϩ) Author contributions: B.P.H.
Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significan... more Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ 54 regulon in a bacterium is that the σ 54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ 54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all σ 54-dependent promoters for the characterization of the σ 54 regulon of Salmonella Typhimurium LT2. Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ 54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ 54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ 54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ 54 binding sites suggest that many of these sites are capable of functioning as σ 54-dependent promoters. Conclusion: Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ 54dependent promoters of the S. Typhimurium LT2 σ 54 regulon under a single growth condition, this approach is likely to be valuable for examining σ 54 regulons in other bacterial species. The S. Typhimurium σ 54 regulon included a high number of intragenic σ 54 binding sites/promoters, suggesting that σ 54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome... more Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome controls peripheral extracellular polysaccharide production and biofilm formation by P. atlantica. High-frequency precise excision of IS492 from epsG requires 5 and 7 bp of flanking DNA, suggesting that IS492 transposition involves a site-specific recombination mechanism. The site specificity of IS492 insertion was examined in P. atlantica and shown to be specific for a 7-bp target, 5-CTTGTTA-3. Characterization of numerous insertion events at the target site in epsG indicated that insertion is also orientation specific. The frequency of IS492 insertion at the epsG target site (2.7 ؋ 10 ؊7 /cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS492 precise excision from the same site. Comparison of insertion sites for IS492 and the highly related ISPtu2 from Pseudoalteromonas tunicata suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS492.
The 54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon ... more The 54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3=-phosphate cyclase (RtcA). Transcription from 54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the 54-dependent promoter of the rsr-yrlBA-rtcBA operon is RtcR. To identify conditions that generate the signal for RtcR activation in S. Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcRdependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion of rtcR or rtcB resulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module, dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon of Escherichia coli (rtcBA) did not stimulate expression of the S. Typhimurium RNA repair operon. Similarly, MMC did not induce expression of the E. coli rtcBA operon, although when expressed in S. Typhimurium, E. coli RtcR responds effectively to the unknown signal(s) generated there by MMC exposure. IMPORTANCE Homologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the 54-dependent RNA repair operon (rsr-yrlBA-rtcBA) of S. Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon components in vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.
Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including trans... more Prokaryotes can adapt to varying environmental conditions by multiple mechanisms, including transient changes in transcription, translation, and posttranslation activities or heritable changes in regulatory or coding DNA sequences. This article addresses mechanisms for high-frequency, stochastic genotypic switching that occurs at DNA regions of increased recombination or mutation, called contingency loci. The heritable DNA rearrangements or mutations that occur at contingency loci pre-adapt a subpopulation of cells for variation in their environment. The systems that are highlighted are the defining examples for control of alternation of gene expression by site-specific DNA inversion, reversible insertion of an IS element, and slipped-strand mispairing at simple sequence DNA repeats.
The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, Rtc... more The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria . In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon.
A regulatory scheme was revealed in which AalR responds to l -Asp and DarR responds to d -Asp, a ... more A regulatory scheme was revealed in which AalR responds to l -Asp and DarR responds to d -Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi .
Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the tra... more Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertio... more Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was address...
Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of t... more Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene famil...
Figure S1. Complete set of 'report cards' for transcription factor binding sites investig... more Figure S1. Complete set of 'report cards' for transcription factor binding sites investigaed in this work. (PDF 3337 kb)
20 Organismal adaptations to environmental stimuli are governed by intracellular signaling molecu... more 20 Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as 21 nucleotide second messengers. Recent studies have identified functional roles for the non-canonical 2 ́,3 ́22 cyclic nucleotide monophosphates (2 ́,3 ́-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli, 23 2 ́,3 ́-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate 24 biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli 25 and Salmonella Typhimurium that are modulated by 2 ́,3 ́-cNMPs through the development of cell26 permeable 2 ́,3 ́-cNMP analogs and a 2 ́,3 ́-cyclic nucleotide phosphodiesterase. Utilization of these 27 chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified 28 pathways regulated by 2 ́,3 ́-cNMPs, including flagellar motility and biofilm formation, and by 29 oligoribonucleotides with 3’-terminal 2 ́,3...
Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significan... more Background: Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ regulon in a bacterium is that the σ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ-dependent promoters for the characterization of the σ regulon of Salmonella Typhimurium LT2. Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcr...
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Papers by Anna Karls