Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene

Predicting non-coding RNA genes in Escherichia coli with boosted genetic programming

Several methods exist for predicting non-coding RNA (ncRNA) genes in Escherichia coli (E.coli). In addition to about sixty known ncRNA genes excluding tRNAs and rRNAs, various methods have predicted more than thousand ncRNA genes, but only 95 of these candidates were confirmed by more than one study. Here, we introduce a new method that uses automatic discovery of sequence patterns to predict ncRNA genes. The method predicts 135 novel candidates. In addition, the method predicts 152 genes that overlap with predictions in the literature. We test sixteen predictions experimentally, and show that twelve of these are actual ncRNA transcripts. Six of the twelve verified candidates were novel predictions. The relatively high confirmation rate indicates that many of the untested novel predictions are also ncRNAs, and we therefore speculate that E.coli contains more ncRNA genes than previously estimated

Single Transmembrane Peptide DinQ Modulates Membrane-Dependent Activities

The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membranedependent activities such as nucleoid compaction and recombination. Copyright: 2013 Weel-Sneve et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Tiling array study of MNNG treated Escherichia coli reveals a widespread transcriptional response

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is known to trigger the adaptive response by inducing the ada-regulon – consisting of three DNA repair enzymes Ada, AlkB, AlkA and the enigmatic AidB. We have applied custom designed tiling arrays to study transcriptional changes in Escherichia coli following a MNNG challenge. Along with the expected upregulation of the adaptive response genes (ada, alkA and alkB), we identified a number of differentially expressed transcripts, both novel and annotated. This indicates a wider regulatory response than previously documented. There were 250 differentially-expressed and 2275 similarly-expressed unannotated transcripts. We found novel upregulation of several stress-induced transcripts, including the SOS inducible genes recN and tisAB, indicating a novel role for these genes in alkylation repair. Furthermore, the ada-regulon A and B boxes were found to be insufficient to explain the regulation of the adaptive response genes after MNNG exposure, suggesting that additional regulatory elements must be involved

Tiling Array Analysis of UV Treated Escherichia coli Predicts Novel Differentially Expressed Small Peptides

Background Despite comprehensive investigation, the Escherichia coli SOS response system is not yet fully understood. We have applied custom designed whole genome tiling arrays to measure UV invoked transcriptional changes in E. coli. This study provides a more complete insight into the transcriptome and the UV irradiation response of this microorganism. Results We detected a number of novel differentially expressed transcripts in addition to the expected SOS response genes (such as sulA, recN, uvrA, lexA, umuC and umuD) in the UV treated cells. Several of the differentially expressed transcripts might play important roles in regulation of the cellular response to UV damage. We have predicted 23 novel small peptides from our set of detected non-gene transcripts. Further, three of the predicted peptides were cloned into protein expression vectors to test the biological activity. All three constructs expressed the predicted peptides, in which two of them were highly toxic to the cell. Additionally, a remarkably high overlap with previously in-silico predicted non-coding RNAs (ncRNAs) was detected. Generally we detected a far higher transcriptional activity than the annotation suggests, and these findings correspond with previous transcription mappings from E. coli and other organisms. Conclusions Here we demonstrate that the E. coli transcriptome consists of far more transcripts than the present annotation suggests, of which many transcripts seem important to the bacterial stress response. Sequence alignment of promoter regions suggest novel regulatory consensus sequences for some of the upregulated genes. Finally, several of the novel transcripts identified in this study encode putative small peptides, which are biologically active. © 2010 Thomassen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Genetic interactions between <i>agrB</i> and <i>uvrA</i>/<i>recB</i> and recombination frequency of <i>agrB</i>.

<p>(A) Dilutions of exponentionally growing wild type (AB1157, open square), <i>agrB</i> (BK4148, open diamond), <i>uvrA</i> (BK4180, open triangle), <i>recB</i> (BK4110, black diamond), <i>agrB recB</i> (BK4112, black square), <i>uvrA recB</i> (BK4183, black circle) and <i>agrB uvrA</i> (BK4182, open circle) cells were plated on LB plates and exposed to UV irradiation. Colonies were counted one day after incubation at 37°C. The data are presented as mean of three independent experiments with standard deviation. (B) Recombination frequency of <i>uvrA</i> (BK4180), <i>dinQ</i> (BK4040) and <i>agrB</i> (BK4043/BK4148) determined by Hfr conjugation assays. The data are presented as mean of three independent experiments with standard deviation.</p

DinQ translation and localization.

<p>(A) Translation of putative DinQ peptides I–V expressed from pET28b(+) plasmid constructs (lanes 1–6) and PCR products corresponding to <i>dinQ-a, -b and -d</i> mRNAs (lane 7–10) were analyzed with coupled <i>in vitro</i> transcription/translation kits from Promega. The <i>E. coli</i> T7 S30 extract system for circular DNA was used to analyze DinQ expression from plasmid constructs while S30 extract system for linear templates was used to analyze PCR products (Promega). Labelling was carried out with [¹⁴C]-Leucine. (B) Western analysis of FLAG-tagged endogenous DinQ expression. Protein extracts from UV exposed (20 J/m²) control cells (BK4044) mixed with plasmid pCR2.1-DinQ-3×-FLAG in lane#1 (pos ctrl), wt (BK5300) in lane#2 (neg ctrl), UV exposed (50 J/m²) <i>ΔagrB</i> DinQ-3×FLAG (BK5372) in lane#3, UV exposed (50 J/m²) wt DinQ-3×FLAG (BK5370) in lane#4, unexposed <i>ΔagrB</i> DinQ-3×FLAG (BK5372) in lane#5, unexposed wt DinQ-3×FLAG (BK5370) in lane#6 was resolved by SDS-PAGE and analyzed by western blotting using Monoclonal ANTI-FLAG M2-Alkaline Phosphatase antibody (SIGMA). Gel migration was monitored relative to SeeBlue Plus2 prestained standard (Invitrogen) in kDa. Detection was carried out by NBT/BCIP color development substrates. The intensities of the DinQ bands was analyzed in three independent western blots with the program ImageJ (Rasband,W.S. and ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA) and normalized against a cross reacting higher molecular weight protein band (not shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003260#pgen-1003260-g004" target="_blank">Figure 4B</a>). A cross reacting low molecular protein band of unknown origin is present in all lanes. (C) Serially diluted (10⁻¹–10⁻⁵cells ml⁻¹) log phase cultures of wt (BK5300), <i>ΔagrB</i> (BK5342), wt <i>dinQ-</i>K4stop (BK5350), <i>ΔagrB dinQ</i>-K4stop (BK5352), wt <i>dinQ</i>-A108T-C112G-A115T (BK5360) and <i>ΔagrB dinQ</i>-A108T-C112G-A115T (BK5362) were spotted onto LB plates and exposed to UV (0 J/m² and 20 J/m²). Pictures were taken one day after incubation at 37°C. (D) Subcellular localization of DinQ in wt (ER2566) cells carrying plasmid pET28b(+)-3×FLAG-DinQ V. Subcellular fractions was resolved by SDS-PAGE and analyzed by western blotting using antibodies against TolC, Lep and FLAG. Lep and TolC detected inner- and outer membrane proteins, respectively. T: total protein; C: cytoplasmic fraction; IM: inner membrane fraction; OM: outer membrane fraction. (E) DinQ amino acid sequence with predicted secondary structure elements (H = helix, ‘-’ = other) and corresponding reliability index (range 0–9). (F) 3D modelling of DinQ as a regular α-helix embedded in a lipid membrane. Polar patch formed by residues Glu17, Arg20 and Gln24 encircled by dashed ellipse.</p