Metal-Responsive Transcription Factors Co-Regulate Anti-Sigma Factor (Rsd) and Ribosome Dimerization Factor Expression.

Bacteria exposed to stress survive by regulating the expression of several genes at the transcriptional and translational levels. For instance, in Escherichia coli, when growth is arrested in response to stress, such as nutrient starvation, the anti-sigma factor Rsd is expressed to inactivate the global regulator RpoD and activate the sigma factor RpoS. However, ribosome modulation factor (RMF) expressed in response to growth arrest binds to 70S ribosomes to form inactive 100S ribosomes and inhibit translational activity. Moreover, stress due to fluctuations in the concentration of metal ions essential for various intracellular pathways is regulated by a homeostatic mechanism involving metal-responsive transcription factors (TFs). Therefore, in this study, we examined the binding of a few metal-responsive TFs to the promoter regions of rsd and rmf through promoter-specific TF screening and studied the effects of these TFs on the expression of rsd and rmf in each TF gene-deficient E. coli strain through quantitative PCR, Western blot imaging, and 100S ribosome formation analysis. Our results suggest that several metal-responsive TFs (CueR, Fur, KdpE, MntR, NhaR, PhoP, ZntR, and ZraR) and metal ions (Cu2+, Fe2+, K+, Mn2+, Na+, Mg2+, and Zn2+) influence rsd and rmf gene expression while regulating transcriptional and translational activities.

Genomic SELEX Reveals Pervasive Role of the Flagella Master Regulator FlhDC in Carbon Metabolism.

Flagella are vital bacterial organs that allow microorganisms to move to favorable environments. However, their construction and operation consume a large amount of energy. The master regulator FlhDC mediates all flagellum-forming genes in E. coli through a transcriptional regulatory cascade, the details of which remain elusive. In this study, we attempted to uncover a direct set of target genes in vitro using gSELEX-chip screening to re-examine the role of FlhDC in the entire E. coli genome regulatory network. We identified novel target genes involved in the sugar utilization phosphotransferase system, sugar catabolic pathway of glycolysis, and other carbon source metabolic pathways in addition to the known flagella formation target genes. Examining FlhDC transcriptional regulation in vitro and in vivo and its effects on sugar consumption and cell growth suggested that FlhDC activates these new targets. Based on these results, we proposed that the flagella master transcriptional regulator FlhDC acts in the activation of a set of flagella-forming genes, sugar utilization, and carbon source catabolic pathways to provide coordinated regulation between flagella formation, operation and energy production.

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid-liquid phase separation and liquid-to-solid transition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid-liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.

Regulatory role of CsuR (YiaU) in determination of cell surface properties of Escherichia coli K-12.

Genomic SELEX screening was performed to identify the binding sites of YiaU, an uncharacterized LysR family transcription factor, on the Escherichia coli K-12 genome. Five high-affinity binding targets of YiaU were identified, all of which were involved in the structures of the bacterial cell surface such as outer and inner membrane proteins, and lipopolysaccharides. Detailed in vitro and in vivo analyses suggest that YiaU activates these target genes. To gain insight into the effects of YiaU in vivo on physiological properties, we used phenotype microarrays, biofilm screening assays and the sensitivity against serum complement analysed using a yiaU deletion mutant or YiaU expression strain. Together, these results suggest that the YiaU regulon confers resistance to some antibiotics, and increases biofilm formation and complement sensitivity. We propose renaming YiaU as CsuR (regulator of cell surface).

Transcription Factor SrsR (YgfI) Is a Novel Regulator for the Stress-Response Genes in Stationary Phase in Escherichia coli K-12.

Understanding the functional information of all genes and the biological mechanism based on the comprehensive genome regulation mechanism is an important task in life science. YgfI is an uncharacterized LysR family transcription factor in Escherichia coli. To identify the function of YgfI, the genomic SELEX (gSELEX) screening was performed for YgfI regulation targets on the E. coli genome. In addition, regulatory and phenotypic analyses were performed. A total of 10 loci on the E. coli genome were identified as the regulatory targets of YgfI with the YgfI binding activity. These predicted YgfI target genes were involved in biofilm formation, hydrogen peroxide resistance, and antibiotic resistance, many of which were expressed in the stationary phase. The TCAGATTTTGC sequence was identified as an YgfI box in in vitro gel shift assay and DNase-I footprinting assays. RT-qPCR analysis in vivo revealed that the expression of YgfI increased in the stationary phase. Physiological analyses suggested the participation of YgfI in biofilm formation and an increase in the tolerability against hydrogen peroxide. In summary, we propose to rename ygfI as srsR (a stress-response regulator in stationary phase).

Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12.

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study 'promoter-specific transcription-factor' (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

Single-target regulators form a minor group of transcription factors in Escherichia coli K-12.

The identification of regulatory targets of all TFs is critical for understanding the entire network of the genome regulation. The lac regulon of Escherichia coli K-12 W3110 is composed of the lacZYA operon and its repressor lacI gene, and has long been recognized as the seminal model of transcription regulation in bacteria with only one highly preferred target. After the Genomic SELEX screening in vitro of more than 200 transcription factors (TFs) from E. coli K-12, however, we found that most TFs regulate multiple target genes. With respect to the number of regulatory targets, a total of these 200 E. coli TFs form a hierarchy ranging from a single target to as many as 1000 targets. Here we focus a total of 13 single-target TFs, 9 known TFs (BetI, KdpE, LacI, MarR, NanR, RpiR, TorR, UlaR and UxuR) and 4 uncharacterized TFs (YagI, YbaO, YbiH and YeaM), altogether forming only a minor group of TFs in E. coli. These single-target TFs were classified into three groups based on their functional regulation.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Regulatory role of CsqR (YihW) in transcription of the genes for catabolism of the anionic sugar sulfoquinovose (SQ) in Escherichia coli K-12.

The binding sites of YihW, an uncharacterized DeoR-family transcription factor (TF) of Escherichia coli K-12, were identified using Genomic SELEX screening at two closely located sites, one inside the spacer between the bidirectional transcription units comprising the yihUTS operon and the yihV gene, and another one upstream of the yihW gene itself. Recently the YihUTS and YihV proteins were identified as catalysing the catabolism of sulfoquinovose (SQ), a hydrolysis product of sulfoquinovosyl diacylglycerol (SQDG) derived from plants and other photosynthetic organisms. Gel shift assay in vitro and reporter assay in vivo indicated that YihW functions as a repressor for all three transcription units. De-repression of the yih operons was found to be under the control of SQ as inducer, but not of lactose, glucose or galactose. Furthermore, a mode of its cooperative DNA binding was suggested for YihW by atomic force microscopy. Hence, as a regulator of the catabolism of SQ, we renamed YihW as CsqR.

Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors.

Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/).

Cross-regulation between two common ancestral response regulators, HprR and CusR, in Escherichia coli.

The uncharacterized two-component system YedVW of Escherichia coli is involved in stress response to hydrogen peroxide. To identify the H2O2-sensing role of YedV, a set of single Cys-to-Ala substitution mutants were constructed. One particular mutant with C165A substitution in the membrane domain rendered YedV inactive in H2O2-dependent transcription of its regulatory target hiuH. We then proposed to rename YedVW to HprSR (hydrogen peroxide response sensor/regulator). One unique characteristic of HprR is the overlapping of its recognition sequence with that of the Cu(II)-response two-component system regulator CusR. Towards understanding this unique regulation system, in this study we analysed the interplay between HprR and CusR with respect to transcription of hiuH, a regulatory target of HprR, and cusC, a target of CusR. Under low protein concentrations in vitro and in vivo, two regulators recognize and transcribe both hiuH and cusC promoters, albeit at different efficiency, apparently in a collaborative fashion. This is a new type of transcription regulation of the common target genes in response to different external signals. Upon increase in protein concentrations, however, HprR and CusR compete with each other in transcription of the common targets, thereby exhibiting a competitive interplay.

TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation.

Growth and differentiation of the neurites depends on long-distance transport of a specific set of mRNAs to restricted area and their local translation. Here, we found that a TAR DNA-binding protein of 43 kDa in size (TDP-43) plays an essential role in intracellular transport of mRNA. For identification of target RNAs recognized by TDP-43, we purified TDP-43 in soluble dimer form and subjected to in vitro systematic evolution of ligands by exponential enrichment (SELEX) screening. All the TDP-43-bound RNAs were found to contain G-quadruplex (G4). Using a double-fluorescent probe system, G4-containing RNAs were found to be transported, together with TDP-43, into the distal neurites. Two lines of evidence indicated that loss of function of TDP-43 results in the neurodegenerative disorder: (i) amyotrophic lateral sclerosis (ALS)-linked mutant TDP-43M337V lacks the activity of binding and transport of G4-containing mRNAs; and (ii) RNA containing G4-forming GGGGCC repeat expansion from the ALS-linked C9orf72 gene absorbs TDP-43, thereby reducing the intracellular pool of functional TDP-43. Taken together, we propose that TDP-43 within neurons plays an essential role of mRNA transport into distal neurites for local translation, and thus, dysfunctions of TDP-43 cause neural diseases such as ALS and frontotemporal lobar degeneration.

Transcription factor DecR (YbaO) controls detoxification of L-cysteine in Escherichia coli.

YbaO is an uncharacterized AsnC-family transcription factor of Escherichia coli. In both Salmonella enterica and Pantoea ananatis, YbaO homologues were identified to regulate the adjacent gene encoding cysteine desulfhydrase for detoxification of cysteine. Using the genomic SELEX (systematic evolution of ligands by exponential enrichment) screening system, we identified the yhaOM operon, located far from the ybaO gene on the E. coli genome, as a single regulatory target of YbaO. In both gel shift assay in vitro and reporter and Northern blot assays in vivo, YbaO was found to regulate the yhaOM promoter. The growth of mutants lacking either ybaO or its targets yhaOM was delayed in the presence of cysteine, indicating involvement of these genes in cysteine detoxification. In the major pathway of cysteine degradation, hydrogen sulfide is produced in wild-type E. coli, but its production was not observed in each of the ybaO, yhaO and yhaM mutants. The yhaOM promoter was activated in the presence of cysteine, implying the role of cysteine in activation of YbaO. Taken together, we propose that YbaO is the cysteine-sensing transcriptional activator of the yhaOM operon, which is involved in the detoxification of cysteine. We then propose the naming of ybaO as decR (regulator of detoxification of cysteine).

Transcription factor CecR (YbiH) regulates a set of genes affecting the sensitivity of Escherichia coli against cefoperazone and chloramphenicol.

Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity).

Cross talk in promoter recognition between six NarL-family response regulators of Escherichia coli two-component system.

Bacterial two-component system (TCS) is composed of the sensor kinase (SK) and the response regulator (RR). After monitoring an environmental signal or condition, SK activates RR through phosphorylation, ultimately leading to the signal-dependent regulation of genome transcription. In Escherichia coli, a total of more than 30 SK-RR pairs exist, each forming a cognate signal transduction system. Cross talk of the signal transduction takes place at three stages: signal recognition by SK (stage 1); RR phosphorylation by SK (stage 2); and target recognition by RR (stage 3). Previously, we analyzed the stage 2 cross talk between the whole set of E. coli SK-RR pairs and found that the cross talk takes place for certain combinations. As an initial attempt to identify the stage 3 cross talk at the step of target promoter recognition by RR, we analyzed in this study the cross-recognition of target promoters by six NarL-family RRs, EvgA, NarL, NarP, RcsB, UhpA, and UvrY. Results of both in vivo and in vitro studies indicated that the stage 3 cross talk takes place for limited combinations, in particular, including a multifactor-regulated ydeP promoter.

Expanded roles of two-component response regulator OmpR in Escherichia coli: genomic SELEX search for novel regulation targets.

The two-component system (TCS) is a sophisticated bacterial signal transduction system for regulation of genome transcription in response to environmental conditions. The EnvZ-OmpR system is one of the well-characterized TCS of Escherichia coli, responding to changes in environmental osmolality. Regulation has largely focused on the differential expression of two porins, OmpF and OmpC, which transport small molecules across the outer membrane. Recently, it has become apparent that OmpR serves a more global regulatory role and regulates additional targets. To identify the entire set of regulatory targets of OmpR, we performed the genomic SELEX screening of OmpR-binding sites along the E. coli genome. As a result, more than 30 novel genes have been identified to be under the direct control of OmpR. One abundant group includes the genes encoding a variety of membrane-associated transporters that mediate uptake or efflux of small molecules, while another group encodes a set of transcription regulators, raising a concept that OmpR is poised to control a diverse set of responses by altering downstream transcriptional regulators.

Role for σ38 in prolonged survival of Escherichia coli in Drosophila melanogaster.

Bacteria adapt themselves to host environments by altering the pattern of gene expression. The promoter-recognizing subunit σ of bacterial RNA polymerase plays a major role in the selection of genes to be transcribed. Among seven σ factors of Escherichia coli, σ(38) is responsible for the transcription of genes in the stationary phase and under stressful conditions. We found a transient increase of σ(38) when E. coli was injected into the hemocoel of Drosophila melanogaster. The loss of σ(38) made E. coli rapidly eliminated in flies, and flies infected with σ(38)-lacking E. coli stayed alive longer than those infected with the parental strain. This was also observed in fly lines defective in humoral immune responses, but not in flies in which phagocytosis was impaired. The lack of σ(38) did not influence the susceptibility of E. coli to phagocytosis, but made them vulnerable to killing after engulfment. The changes caused by the loss of σ(38) were recovered by the forced expression of σ(38)-encoding rpoS as well as σ(38)-regulated katE and katG coding for enzymes that detoxify reactive oxygen species. These results collectively suggested that σ(38) contributes to the prolonged survival of E. coli in Drosophila by inducing the production of enzymes that protect bacteria from killing in phagocytes. Considering the similarity in the mechanism of innate immunity against invading bacteria between fruit flies and humans, the products of these genes could be new targets for the development of more effective antibacterial remedies.

Regulatory role of transcription factor SutR (YdcN) in sulfur utilization in Escherichia coli.

Sulfur makes up 1 % of the dry mass of bacteria, and it is an abundant element (0.1 %) on earth. Sulfur in the environment is, however, mostly in oxidized forms and inaccessible to living organisms. At present, the entire assimilation pathway of external sulfur to sulfur-containing biomolecules and its regulation in Escherichia coli remain poorly understood, except for the metabolic pathway of cysteine synthesis, the first-step metabolite of sulfur assembly. During the search for regulation targets of uncharacterized transcription factors by Genomic SELEX screening, we found that the hitherto uncharacterized YdcN regulates a set of genes involved in the utilization of sulfur, including the generation of sulfate and its reduction, the synthesis of cysteine, the synthesis of enzymes containing Fe-S as cofactors, and the modification of tRNA with use of sulfur-containing substrates. Taking these findings together, we propose renaming YdcN as SutR (regulator of sulfur utilization).

Cooperative regulation of the common target genes between H2O2-sensing YedVW and Cu²âº-sensing CusSR in Escherichia coli.

YedVW is one of the uncharacterized two-component systems (TCSs) of Escherichia coli. In order to identify the regulation targets of YedVW, we performed genomic SELEX (systematic evolution of ligands by exponential enrichment) screening using phosphorylated YedW and an E. coli DNA library, and identified YedW-binding sites within three intergenic spacers, yedW-hiuH, cyoA-ampG and cusR-cusC, along the E. coli genome. Using a reporter assay system, we found that transcription of hiuH, encoding 5-hydroxyisourate hydrolase, was induced at high concentrations of either Cu(2+) or H2O2. Cu(2+)-dependent expression of hiuH was observed in the yedWV knockout mutant, but was reduced markedly in the cusRS-null mutant. However, H2O2-induced hiuH expression was observed in the cusRS-null mutant, but not in the yedWV-null mutant. Gel mobility shift and DNase I footprinting analyses showed binding of both YedW and CusR to essentially the same sequence within the hiuH promoter region. Taken together, we concluded that YedVW and CusSR formed a unique cooperative TCS pair by recognizing and regulating the same targets, but under different environmental conditions - YedVW played a role in H2O2 response regulation, whilst CusSR played a role in Cu(2+) response regulation.