scholarly journals An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Anna F. Wang Erickson ◽  
Padraig Deighan ◽  
Cinthia P. Garcia ◽  
Robert O. J. Weinzierl ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Sigma Factor ◽  
Coiled Coil ◽  
Sigma Factors ◽  
Alternative Sigma Factor ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Σ Factor

ABSTRACT Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β′ subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σH, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σH-directed gene expression but not in the activity of the stress-response sigma factor σB. Pulldown experiments showed that the mutant RNAP was defective in associating with σH but could still associate with σA and σB. The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β′ coiled coil that is specifically detrimental for interaction with σH. This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor. IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β′ subunit whereby RNAPL257P associates with some σ factors (σA and σB) and enables vegetative cell growth but is defective in utilization of σH and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.

scholarly journals Transcriptional Regulation and Mechanism of SigN (ZpdN), a pBS32-Encoded Sigma Factor in Bacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Aisha T. Burton ◽  
Aaron DeLoughery ◽  
Gene-Wei Li ◽  
Daniel B. Kearns
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Consensus Sequence ◽  
Sigma Factors ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Bona Fide

ABSTRACT Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis also encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to being encoded on the large, low-copy-number plasmid pBS32, which was lost during domestication. DNA damage triggers pBS32 hyperreplication and cell death in a manner that depends on ZpdN, but how ZpdN mediates these effects is unknown. Here, we show that ZpdN is a bona fide sigma factor that can direct RNA polymerase to transcribe ZpdN-dependent genes, and we rename ZpdN SigN accordingly. Rend-seq (end-enriched transcriptome sequencing) analysis was used to determine the SigN regulon on pBS32, and the 5′ ends of transcripts were used to predict the SigN consensus sequence. Finally, we characterize the regulation of SigN itself and show that it is transcribed by at least three promoters: PsigN1, a strong SigA-dependent LexA-repressed promoter; PsigN2, a weak SigA-dependent constitutive promoter; and PsigN3, a SigN-dependent promoter. Thus, in response to DNA damage SigN is derepressed and then experiences positive feedback. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon. IMPORTANCE Sigma factors are utilized by bacteria to control and regulate gene expression. Some sigma factors are activated during times of stress to ensure the survival of the bacterium. Here, we report the presence of a sigma factor that is encoded on a plasmid that leads to cellular death after DNA damage.

scholarly journals DksA and ppGpp Regulate the σS Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Mary E. Girard ◽  
Saumya Gopalkrishnan ◽  
Elicia D. Grace ◽  
Jennifer A. Halliday ◽  
Richard L. Gourse ◽  
...  
Keyword(s):  
Stress Response ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Bacterial Species ◽  
Adaptor Protein ◽  
Large Family ◽  
Alternative Sigma Factor ◽  
Term Survival ◽  
Content Type

ABSTRACT σS is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σS general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σS levels transcriptionally, translationally, and posttranslationally in Escherichia coli. It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σS synthesis, thus playing an important role in activation of the σS stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σS levels. In addition, based on effects of DksAN88I, a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σS activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σS and also increase transcription from other promoters, including PdsrA and PiraP. By improving the translation and stabilization of σS, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σS. Understanding the factors governing σS activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σS levels.

scholarly journals Role of autoregulation and relative synthesis of operon partners in alternative sigma factor networks

2015 ◽  
Author(s):  
Jatin Narula ◽  
Abhinav Tiwari ◽  
Oleg A. Igoshin
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Negative Feedback ◽  
Sigma Factor ◽  
Critical Role ◽  
Sigma Factors ◽  
Alternative Sigma Factor ◽  
Negative Feedback Regulation ◽  
Alternative Sigma Factors

SummaryDespite the central role of alternative sigma factors in bacterial stress response and virulence their regulation remains incompletely understood. Here we investigate one of the best-studied examples of alternative sigma factors: the σBnetwork that controls the general stress response ofBacillus subtilisto uncover widely relevant general design principles that describe the structure-function relationship of alternative sigma factor regulatory networks. We show that the relative stoichiometry of the synthesis rates of σB, its anti-sigma factor RsbW and the anti-anti-sigma factor RsbV plays a critical role in shaping the network behavior by forcing the σBnetwork to function as an ultrasensitive negative feedback loop. We further demonstrate how this negative feedback regulation insulates alternative sigma factor activity from competition with the housekeeping sigma factor for RNA polymerase and allows multiple stress sigma factors to function simultaneously with little competitive interference.Major Subject Areas:Computational and systems biology, Microbiology & Infectious diseaseResearch Organism:Bacillus subtilis

scholarly journals The evolutionary impact of Intragenic FliA Promoters in Proteobacteria

2017 ◽  
Author(s):  
Devon M. Fitzgerald ◽  
Carol Smith ◽  
Pascal Lapierre ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Protein Coding ◽  
Alternative Sigma Factors ◽  
Genome Scale ◽  
The Impact ◽  
Flagellar Genes

ABSTRACTRecent work has revealed that large numbers of promoters in bacteria are located inside genes. In contrast, almost all studies of transcription have focused on promoters upstream of genes. Bacterial promoters are recognized by Sigma factors that associate with initiating RNA polymerase. In Escherichia coli, one Sigma factor recognizes the majority of promoters, and six “alternative” Sigma factors recognize specific subsets of promoters. One of these alternative Sigma factors, FliA (σ28), recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, reveal that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription is a conserved phenomenon, but that the specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved, and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence protein evolution in bacteria, and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.AUTHOR SUMMARYRecent genome-scale studies of bacterial transcription have revealed thousands of promoters inside genes. In a few cases, these promoters have been shown to transcribe functional RNAs. However, it is unclear whether most intragenic promoters have important biological function. Similarly, there are likely to be thousands of intragenic binding sites for transcription factors, but very few have been functionally characterized. Moreover, it is unclear what impact intragenic promoters and transcription factor binding sites have on evolution of the overlapping genes. In this study, we focus on FliA, a broadly conserved Sigma factor that is responsible for initiating transcription of many flagellar genes. We previously showed that FliA directs RNA polymerase to ~50 genomic sites in Escherichia coli. In our current study, we show that while most intragenic FliA promoters are actively transcribed, very few are conserved in other species. This suggests that most FliA promoters are not functional. Nonetheless, one intragenic FliA promoter is highly conserved, and we show that this promoter constrains evolution of the overlapping protein-coding gene. Given the enormous number of regulatory DNA sites within genes, we propose that the evolution of many genes is constrained by these elements.

scholarly journals Interactions between DksA and Stress-Responsive Alternative Sigma Factors Control Inorganic Polyphosphate Accumulation in Escherichia coli

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Michael J. Gray
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Nutrient Limitation ◽  
Protein Function ◽  
Regulatory Protein ◽  
Sigma Factors ◽  
Inorganic Polyphosphate ◽  
Content Type ◽  
Alternative Sigma Factors

ABSTRACT Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coli. IMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.

scholarly journals Growth Phase-Dependent Regulation of the Extracytoplasmic Stress Factor, σE, by Guanosine 3′,5′-Bispyrophosphate (ppGpp)

2006 ◽  
Vol 188 (13) ◽  
pp. 4627-4634 ◽  
Author(s):  
Alessandra Costanzo ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Growth Phase ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Alternative Sigma Factor ◽  
Dependent Manner ◽  
Alternative Sigma Factors ◽  
Promoter Recognition ◽  
Sigma Subunit

ABSTRACT The sigma subunit of procaryotic RNA polymerases is responsible for specific promoter recognition and transcription initiation. In addition to the major sigma factor, σ70, in Escherichia coli, which directs most of the transcription in the cell, bacteria possess multiple, alternative sigma factors that direct RNA polymerase to distinct sets of promoters in response to environmental signals. By activating an alternative sigma factor, gene expression can be rapidly reprogrammed to meet the needs of the cell as the environment changes. Sigma factors are subject to multiple levels of regulation that control their levels and activities. The alternative sigma factor σE in Escherichia coli is induced in response to extracytoplasmic stress. Here we demonstrate that σE can also respond to signals other than extracytoplasmic stress. σE activity increases in a growth phase-dependent manner as a culture enters stationary phase. The signaling pathway that activates σE during entry into stationary phase is dependent upon the alarmone guanosine 3′,5′-bispyrophosphate (ppGpp) and is distinct from the pathway that signals extracytoplasmic stress. ppGpp is the first cytoplasmic factor shown to control σE activity, demonstrating that σE can respond to internal signals as well as signals originating in the cell envelope. ppGpp is a general signal of starvation stress and is also required for activation of the σS and σ54 alternative sigma factors upon entry into stationary phase, suggesting that this is a key mechanism by which alternative sigma factors can be activated in concert to provide a coordinated response to nutritional stress.

scholarly journals Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502

2013 ◽  
Vol 79 (12) ◽  
pp. 3867-3869 ◽  
Author(s):  
Elias Dahlsten ◽  
David Kirk ◽  
Miia Lindström ◽  
Hannu Korkeala
Keyword(s):  
Stress Tolerance ◽  
Sigma Factor ◽  
Clostridium Botulinum ◽  
Alternative Sigma Factor ◽  
Strain Atcc ◽  
Content Type ◽  
Osmotic Stress Tolerance ◽  
Group I ◽  
Temperature Downshift

ABSTRACTThe role of the alternative sigma factor SigK in cold and osmotic stress tolerance ofClostridium botulinumATCC 3502 was demonstrated by induction ofsigKafter temperature downshift and exposure to hyperosmotic conditions and by impaired growth of thesigKmutants under the respective conditions.

scholarly journals A Tripartite, Hierarchical Sigma Factor Cascade Promotes Hormogonium Development in the Filamentous Cyanobacterium Nostoc punctiforme

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Alfonso Gonzalez ◽  
Kelsey W. Riley ◽  
Thomas V. Harwood ◽  
Esthefani G. Zuniga ◽  
Douglas D. Risser
Keyword(s):  
Sigma Factor ◽  
Dominant Role ◽  
Sigma Factors ◽  
Oxygenic Photosynthesis ◽  
Model Organisms ◽  
Filamentous Cyanobacterium ◽  
Nostoc Punctiforme ◽  
Filamentous Cyanobacteria ◽  
Content Type ◽  
Nitrogen Cycles

ABSTRACT Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and frequently, nitrogen fixation as well. As a result, they contribute substantially to global primary production and nitrogen cycles. Furthermore, the multicellular filamentous cyanobacteria in taxonomic subsections IV and V are developmentally complex, exhibiting an array of differentiated cell types and filaments, including motile hormogonia, making them valuable model organisms for studying development. To investigate the role of sigma factors in the gene regulatory network (GRN) controlling hormogonium development, a combination of genetic, immunological, and time-resolved transcriptomic analyses were conducted in the model filamentous cyanobacterium Nostoc punctiforme, which, unlike other common model cyanobacteria, retains the developmental complexity of field isolates. The results support a model where the hormogonium GRN is driven by a hierarchal sigma factor cascade, with sigJ activating the expression of both sigC and sigF, as well as a substantial portion of additional hormogonium-specific genes, including those driving changes to cellular architecture. In turn, sigC regulates smaller subsets of genes for several processes, plays a dominant role in promoting reductive cell division, and may also both positively and negatively regulate sigJ to reinforce the developmental program and coordinate the timing of gene expression, respectively. In contrast, the sigF regulon is extremely limited. Among genes with characterized roles in hormogonium development, only pilA shows stringent sigF dependence. For sigJ-dependent genes, a putative consensus promoter was also identified, consisting primarily of a highly conserved extended −10 region, here designated a J-Box, which is widely distributed among diverse members of the cyanobacterial lineage. IMPORTANCE Cyanobacteria are integral to global carbon and nitrogen cycles, and their metabolic capacity coupled with their ease of genetic manipulation make them attractive platforms for applications such as biomaterial and biofertilizer production. Achieving these goals will likely require a detailed understanding and precise rewiring of these organisms’ GRNs. The complex phenotypic plasticity of filamentous cyanobacteria has also made them valuable models of prokaryotic development. However, current research has been limited by focusing primarily on a handful of model strains which fail to reflect the phenotypes of field counterparts, potentially limiting biotechnological advances and a more comprehensive understanding of developmental complexity. Here, using Nostoc punctiforme, a model filamentous cyanobacterium that retains the developmental range of wild isolates, we define previously unknown definitive roles for a trio of sigma factors during hormogonium development. These findings substantially advance our understanding of cyanobacterial development and gene regulation and could be leveraged for future applications.

scholarly journals Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation

2019 ◽  
Vol 71 (3) ◽  
pp. 1029-1038 ◽  
Author(s):  
Lauren A Macadlo ◽  
Iskander M Ibrahim ◽  
Sujith Puthiyaveetil
Keyword(s):  
Rna Polymerase ◽  
Gene Transcription ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Sigma Factors ◽  
Transcriptional Level ◽  
Gene Promoters ◽  
Chloroplast Genes ◽  
Specific Subset ◽  
Bacterial Rna

Abstract Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

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