scholarly journals Transcriptional Regulation and Mechanism of SigN (ZpdN), a pBS32 encoded Sigma Factor

2019 ◽  
Author(s):  
Aisha T. Burton ◽  
Aaron DeLoughery ◽  
Gene-Wei Li ◽  
Daniel B. Kearns
Keyword(s):  
Dna Damage ◽  
Sigma Factor ◽  
Consensus Sequence ◽  
Sigma Factors ◽  
Dependent Manner ◽  
Regulate Gene Expression ◽  
Alternative Sigma Factors ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Bona Fide

ABSTRACTLaboratory strains of Bacillus subtilis encodes as many as 16 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 it being encoded on the large, low copy number plasmid pBS32 that was lost during domestication. DNA damage triggers pBS32 hyper-replication and cell death in a manner that depends on ZpdN but how ZpdN mediates these effects was 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 to SigN accordingly. Rend-seq 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 LexA is derepressed, SigN is expressed 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.IMPORTANCESigma factors are utilized by bacteria to control and regulate gene expression. Extra cytoplasmic function 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 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 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 The Single Extracytoplasmic-Function Sigma Factor of Xylella fastidiosa Is Involved in the Heat Shock Response and Presents an Unusual Regulatory Mechanism

2006 ◽  
Vol 189 (2) ◽  
pp. 551-560 ◽  
Author(s):  
José F. da Silva Neto ◽  
Tie Koide ◽  
Suely L. Gomes ◽  
Marilis V. Marques
Keyword(s):  
Heat Shock ◽  
Sigma Factor ◽  
Xylella Fastidiosa ◽  
Expression Profiles ◽  
Consensus Sequence ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Sigma Factors ◽  
Bacterial Cells ◽  
Ecf Sigma Factor

ABSTRACT Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa σE regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 σE-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative σE-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified σE-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.

scholarly journals Three Asparagine Synthetase Genes ofBacillus subtilis

1999 ◽  
Vol 181 (19) ◽  
pp. 6081-6091 ◽  
Author(s):  
Ken-Ichi Yoshida ◽  
Yasutaro Fujita ◽  
S. Dusko Ehrlich
Keyword(s):  
Stationary Phase ◽  
Exponential Growth ◽  
Minimal Medium ◽  
Slow Growth ◽  
Sigma Factors ◽  
Asparagine Synthetase ◽  
Sporulation Medium ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Sigma E

ABSTRACT Three asparagine synthetase genes, asnB,asnH, and asnO (yisO), were predicted from the sequence of the Bacillus subtilisgenome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression ofasnB was detected only during exponential growth, that ofasnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnOwas seen only later in sporulation. In a minimal medium, bothasnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression ofasnH was partially repressed. Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprisingyxbB, yxbA, yxnB, asnH, and yxaM. The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of anEscherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In B. subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion ofasnB led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B. subtilismight have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation.

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 Effects of DNA Topology on Transcription from rRNA Promoters in Bacillus subtilis

2021 ◽  
Vol 9 (1) ◽  
pp. 87
Author(s):  
Petra Sudzinová ◽  
Milada Kambová ◽  
Olga Ramaniuk ◽  
Martin Benda ◽  
Hana Šanderová ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Additional Data ◽  
Dna Supercoiling ◽  
Dna Topology ◽  
Sigma Factors ◽  
Dependent Manner ◽  
Dna Relaxation ◽  
Cellular Processes ◽  
Alternative Sigma Factors

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.

scholarly journals Alternative Sigma Factors and Their Roles in Bacterial Virulence

2005 ◽  
Vol 69 (4) ◽  
pp. 527-543 ◽  
Author(s):  
Mark J. Kazmierczak ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Sigma Factor ◽  
Transcription Initiation ◽  
Virulence Genes ◽  
Sigma Factors ◽  
Bacterial Virulence ◽  
Bacterial Survival ◽  
Host Organism ◽  
Alternative Sigma Factors ◽  
Promoter Recognition ◽  
Dna Strand

SUMMARY Sigma factors provide promoter recognition specificity to RNA polymerase holoenzyme, contribute to DNA strand separation, and then dissociate from the core enzyme following transcription initiation. As the regulon of a single sigma factor can be composed of hundreds of genes, sigma factors can provide effective mechanisms for simultaneously regulating expression of large numbers of prokaryotic genes. One newly emerging field is identification of the specific roles of alternative sigma factors in regulating expression of virulence genes and virulence-associated genes in bacterial pathogens. Virulence genes encode proteins whose functions are essential for the bacterium to effectively establish an infection in a host organism. In contrast, virulence-associated genes can contribute to bacterial survival in the environment and therefore may enhance the capacity of the bacterium to spread to new individuals or to survive passage through a host organism. As alternative sigma factors have been shown to regulate expression of both virulence and virulence-associated genes, these proteins can contribute both directly and indirectly to bacterial virulence. Sigma factors are classified into two structurally unrelated families, the σ70 and the σ54 families. The σ70 family includes primary sigma factors (e.g., Bacillus subtilis σA) as well as related alternative sigma factors; σ54 forms a distinct subfamily of sigma factors referred to as σN in almost all species for which these proteins have been characterized to date. We present several examples of alternative sigma factors that have been shown to contribute to virulence in at least one organism. For each sigma factor, when applicable, examples are drawn from multiple species.

scholarly journals Heme Utilization in Bordetella aviumIs Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor

2001 ◽  
Vol 69 (11) ◽  
pp. 6951-6961 ◽  
Author(s):  
Amy E. Kirby ◽  
Daniel J. Metzger ◽  
Erin R. Murphy ◽  
Terry D. Connell
Keyword(s):  
Sigma Factor ◽  
Receptor Gene ◽  
Regulatory System ◽  
Biological Properties ◽  
Sigma Factors ◽  
Transcriptional Fusion ◽  
Reading Frame ◽  
Alternative Sigma Factors ◽  
Bordetella Avium ◽  
Extracytoplasmic Function Sigma Factor

ABSTRACT Efficient utilization of heme as an iron (Fe) source byBordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized thatrhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuIwas correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal tobhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced intoEscherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. colidemonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of therhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

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.

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