Genetic Composition of the Bacillus subtilis SOS System

ABSTRACT The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5′-CGAACATATGTTCG-3′. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

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Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

Microbiology ◽

10.1099/mic.0.025841-0 ◽

2009 ◽

Vol 155 (5) ◽

pp. 1459-1477 ◽

Cited By ~ 32

Author(s):

Nina Jochmann ◽

Anna-Katharina Kurze ◽

Lisa F. Czaja ◽

Karina Brinkrolf ◽

Iris Brune ◽

...

Keyword(s):

Dna Damage ◽

Corynebacterium Glutamicum ◽

Intergenic Region ◽

Binding Sites ◽

Consensus Sequence ◽

Differentially Expressed ◽

Wild Type ◽

Band Shift ◽

Lexa Binding

The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter–operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

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Identification of Some DNA Damage-Inducible Genes of Mycobacterium tuberculosis: Apparent Lack of Correlation with LexA Binding

Journal of Bacteriology ◽

10.1128/jb.183.15.4459-4467.2001 ◽

2001 ◽

Vol 183 (15) ◽

pp. 4459-4467 ◽

Cited By ~ 49

Author(s):

Patricia C. Brooks ◽

Farahnaz Movahedzadeh ◽

Elaine O. Davis

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

Binding Sites ◽

Alternative Mechanism ◽

Intracellular Pathogens ◽

Apparent Lack ◽

E Coli ◽

Lexa Binding ◽

Inducible Genes

ABSTRACT The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response ofM. tuberculosis to DNA damage. The expression ofrecA, a key component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis. In this study, we have analyzed the expression following DNA damage in M. tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli. While many of these genes were also induced by DNA damage in M. tuberculosis, some were not. In addition, one gene (ruvC) which is not induced by DNA damage in E. coli was induced in M. tuberculosis, a result likely linked to its different transcriptional arrangement in M. tuberculosis. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in E. coli. However, two of the genes which were DNA damage inducible in M. tuberculosis did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M. tuberculosis.

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

mBio ◽

10.1128/mbio.01899-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 4

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.

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Identification of Chlamydia trachomatis Genomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA)

Journal of Bacteriology ◽

10.1128/jb.187.2.443-448.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 443-448 ◽

Cited By ~ 14

Author(s):

Annette Rau ◽

Susan Wyllie ◽

Judy Whittimore ◽

Jane E. Raulston

Keyword(s):

Chlamydia Trachomatis ◽

Divalent Cation ◽

Open Reading Frames ◽

Genomic Sequences ◽

Mobility Shift ◽

Reading Frame ◽

E Coli ◽

Resultant Data ◽

Mobility Shift Assay ◽

Titration Assay

ABSTRACT The Chlamydia trachomatis divalent cation-dependent regulator (DcrA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators. Chlamydial DcrA specifically binds to a consensus Escherichia coli Fur box and is able to complement an E. coli Fur mutant. In this report, the E. coli Fur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized by E. coli Fur. The predictive regulatory regions of 28 C. trachomatis open reading frames contained sequences functionally recognized by E. coli Fur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways. Selected FURTA-positive sequences were subsequently examined for recognition by C. trachomatis DcrA using an electrophoretic mobility shift assay. The resultant data show that C. trachomatis DcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DcrA and the Fur family of regulators.

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Identification of a Repressor for the Two iol Operons Required for Inositol Catabolism in Geobacillus Kaustophilus

10.21203/rs.3.rs-35185/v1 ◽

2020 ◽

Author(s):

Ken-ichi Yoshida ◽

Yusuke Shirae ◽

Ryo Nishimura ◽

Kaho Fukui ◽

Shu Ishikawa

Keyword(s):

Dna Binding ◽

Gene Cluster ◽

Binding Sites ◽

Consensus Sequence ◽

Binding Property ◽

Promoter Regions ◽

Mobility Shift ◽

Geobacillus Kaustophilus ◽

Inositol Dehydrogenase

Abstract BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster consists of two tandem operons induced in the presence of inositol; however, the mechanism underlying the induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding a scyllo-inositol dehydrogenase, and its homolog in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and termed as iolQ in G. kaustophilus.ResultsWhen iolQ was inactivated, not only the myo-inositol dehydrogenase activity in the cell due to the expression of gk1899 but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal His-tag fusion in Escherichia coli and subjected to the in vitro gel mobility shift assay to examine its DNA binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions, and its DNA binding was antagonized by myo-inositol. Moreover, DNase I footprint analyses were conducted to determine the two binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to be a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R = A or G, W = A or T, S = G or C, and Y = C or T).ConclusionIolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

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Changes in DnaA-Dependent Gene Expression Contribute to the Transcriptional and Developmental Response of Bacillus subtilis to Manganese Limitation in Luria-Bertani Medium

Journal of Bacteriology ◽

10.1128/jb.00210-10 ◽

2010 ◽

Vol 192 (15) ◽

pp. 3915-3924 ◽

Cited By ~ 20

Author(s):

Sharon E. Hoover ◽

Weihong Xu ◽

Wenzhong Xiao ◽

William F. Burkholder

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Bacillus Subtilis ◽

Dna Replication ◽

Alkylating Agents ◽

Transcriptional Response ◽

Sos Response ◽

Replication Initiation ◽

Specific Gene ◽

Transcriptional Responses

ABSTRACT The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress.

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CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.05899-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6276-6287 ◽

Cited By ~ 13

Author(s):

Boris R. Belitsky ◽

Abraham L. Sonenshein

Keyword(s):

Bacillus Subtilis ◽

Binding Sites ◽

Consensus Sequence ◽

Regulatory Region ◽

Binding Motif ◽

Downstream Site ◽

Upstream Site ◽

Binding Motifs ◽

Content Type ◽

A Minor

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons inBacillus subtilis. Some of the most strongly repressed targets of CodY, thenupNOPQ(formerly,yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of thenupNgene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-foldnupNrepression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site tonupNregulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.

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YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

Journal of Bacteriology ◽

10.1128/jb.185.18.5380-5390.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5380-5390 ◽

Cited By ~ 17

Author(s):

José M. Salas-Pacheco ◽

Norma Urtiz-Estrada ◽

Guadalupe Martínez-Cadena ◽

Ronald E. Yasbin ◽

Mario Pedraza-Reyes

Keyword(s):

Dna Repair ◽

Bacillus Subtilis ◽

Alkylating Agents ◽

Enzymatic Properties ◽

Mobility Shift ◽

Exonuclease Iii ◽

E Coli ◽

Type Iv ◽

Cell Extracts ◽

Ap Sites

ABSTRACT The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3′-phosphatases. However, the purified protein lacked both 5′-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3′ blocking groups of DNA generated during both spore dormancy and germination.

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The Bacillus subtilis Response Regulator GenedegUIs Positively Regulated by CcpA and by Catabolite-Repressed Synthesis of ClpC

Journal of Bacteriology ◽

10.1128/jb.01881-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 193-201 ◽

Cited By ~ 16

Author(s):

Hiroshi Ishii ◽

Teruo Tanaka ◽

Mitsuo Ogura

Keyword(s):

Bacillus Subtilis ◽

Response Regulator ◽

Consensus Sequence ◽

Protein A ◽

Upstream Region ◽

Sporulation Medium ◽

Mobility Shift ◽

Content Type ◽

Cellular Processes ◽

Mobility Shift Assay

ABSTRACTInBacillus subtilis, the response regulator DegU and its cognate kinase, DegS, constitute a two-component system that regulates many cellular processes, including exoprotease production and genetic competence. Phosphorylated DegU (DegU-P) activates its own promoter and is degraded by the ClpCP protease. We observed induction ofdegUby glucose in sporulation medium. This was abolished in two mutants: theccpA(catabolite control protein A) andclpCdisruptants. Transcription of the promoter of the operon containingclpC(PclpC) decreased in the presence of glucose, and the disruption ofccpAresulted in derepression of PclpC. However, this was not directly mediated by CcpA, because we failed to detect binding of CcpA to PclpC. Glucose decreased the expression ofclpC, leading to low cellular concentrations of the ClpCP protease. Thus,degUis induced through activation of autoregulation by a decrease in ClpCP-dependent proteolysis of DegU-P. An electrophoretic mobility shift assay showed that CcpA bound directly to thedegUupstream region, indicating that CcpA activatesdegUthrough binding. The bound region was narrowed down to 27 bases, which contained acre(catabolite-responsiveelement) sequence with a low match to thecreconsensus sequence. In a footprint analysis, CcpA specifically protected a region containing thecresequence from DNase I digestion. The induction ofdegUby glucose showed complex regulation of thedegUgene.

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Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

Applied and Environmental Microbiology ◽

10.1128/aem.00756-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5435-5446 ◽

Cited By ~ 26

Author(s):

Johannes Sjöholm ◽

Paulo Oliveira ◽

Peter Lindblad

Keyword(s):

Open Reading Frames ◽

Start Codon ◽

Hydrogenase Activity ◽

Promoter Regions ◽

Mobility Shift ◽

Reading Frame ◽

Genes Encoding ◽

Lexa Binding ◽

Bidirectional Hydrogenase ◽

Pcc 7120

ABSTRACT The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and recF. This indicates that, in addition to the bidirectional hydrogenase gene, a number of other genes, including open reading frames connected to DNA replication, recombination, and repair, may be part of the LexA regulatory network in Nostoc sp. strain PCC 7120.

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