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1.
Int J Mol Sci ; 23(11)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35682733

RESUMO

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).


Assuntos
Escherichia coli K12 , Proteínas de Escherichia coli , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
FEMS Microbiol Lett ; 366(2)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30624637

RESUMO

When the rate of production of metabolites in bacteria exceeds the amounts needed for cell growth, excess metabolites are secreted into the extracellular environment. Upon entry into poor nutrient conditions, overflowed exometabolites are reused to continue cell growth and survival. At present, however, the genetic system for utilization of exometabolites is poorly understood even for the best-characterized model prokaryote Escherichia coli. A two-component system YpdAB of E. coli K-12 was predicted to participate in regulation of this process, and the yhjX gene encoding the MFS-family transporter with an as yet unidentified function was identified as a single regulatory target of YpdB. Using gSELEX screening in vitro, however, we have identified up to eight regulatory targets, including the yhjX gene. The predicted regulatory targets were all confirmed to be under the direct control of YpdB by gel shift assay in vitro and reporter assay in vivo. For induction of YpdAB function, the major exometabolite pyruvate in growing E. coli K-12 was identified as the inducer. We then propose to rename YpdAB as PyrSR (regulator of pyruvate reutilization). One unique feature of PyrSR is its cross-talk with another pyruvate-sensing BtsSR at the TCS stage 1 for fine-tuning of pyruvate reutilization.


Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Ácido Pirúvico/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases/genética , Fatores de Transcrição/genética
3.
Trends Biotechnol ; 35(10): 983-996, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28739220

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated manipulation of genomic information is becoming more versatile by combining nuclease-deficient CRISPR systems with a wide variety of effectors including base-editing deaminases, transcriptional regulators, and epigenetic modifiers. The programmable binding ability of CRISPR systems is essential when the systems are employed as targeting domains to recruit the effectors to specific genomic loci. The discovery of a variety of Cas9 orthologs and engineered variants enables high-fidelity genome editing and a wider selection of genomic targets, and CRISPR-mediated deaminases enable more precise and predictable genome editing compared with CRISPR nuclease-based editing. Finally, combining transcriptional regulators with CRISPR systems can control expression of specific genes in a genome. Some applications and future challenges of CRISPR-derived tools are also discussed.


Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Endonucleases , Edição de Genes/métodos , Loci Gênicos , Engenharia de Proteínas/métodos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Endonucleases/química , Endonucleases/genética , Humanos
4.
Science ; 353(6301): 819-22, 2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-27540174

RESUMO

Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Sintéticos , Código Genético/fisiologia , Genoma Bacteriano , Genes Essenciais , Genes Letais , Código Genético/genética , Engenharia Genética , Fenótipo , Biossíntese de Proteínas/genética
5.
mBio ; 5(4): e01442-14, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-25006232

RESUMO

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.


Assuntos
Escherichia coli/genética , Óperon/genética , Análise de Sequência de RNA/métodos , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica/genética , Transcriptoma/genética
7.
Biophys J ; 105(4): 1037-44, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23972855

RESUMO

The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30-60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/metabolismo , Bacteriófago T4 , Proliferação de Células/efeitos dos fármacos , Fenômenos Químicos , Espaço Intracelular/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , Poliaminas/metabolismo , Ligação Proteica , Espermidina/farmacologia
8.
Nucleic Acids Res ; 41(2): 746-53, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23180762

RESUMO

Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan-DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , DNA/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestrutura , Anaerobiose , Cromossomos Bacterianos , DNA/metabolismo , DNA/ultraestrutura , Escherichia coli/genética , Cloreto de Magnésio/química
9.
Genes Cells ; 15(11): 1111-22, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20964794

RESUMO

Laboratory cultures of a single species of bacteria harboring the same genetic background include heterogeneous cell populations, each differing in apparent morphology and physiology, as found in natural environments. To get insights into difference in the genome expression between individual cells, we constructed various types of the cell chip for monitoring the growth and fate of individual bacterial cells. Immobilization of portions of Escherichia coli culture within these cell chips was established after raising the local temperature in the presence of poly-(N-isopropylacrylamide) (PNIPAAm). The newly developed cell-chip system allows the investigation of activity and regulation of green fluorescent protein (GFP)-fused promoter in single live-bacterial cells for prolonged time under controlled culture conditions. Using this single-cell observation system, we succeeded, for the first time, the real-time single-cell assay of promoter activity of the E. coli gcl gene encoding glyoxylate carboligase as a model system, and the kinetics of gcl induction by an effector glyoxylate. Marked heterogeneity was found in the expression level of the gcl promoter. The heterogeneity in gcl promoter activity was, however, confirmed by Flow cytometry of suspension cultures. Our success provides an experimental system for the increased demand of single-cell biology in bacterial studies.


Assuntos
Bioensaio/métodos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Acrilamidas/química , Técnicas Bacteriológicas , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Repressoras/genética
10.
Nucleic Acids Res ; 38(11): 3605-18, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20156994

RESUMO

A systematic search was performed for DNA-binding sequences of YgiP, an uncharacterized transcription factor of Escherichia coli, by using the Genomic SELEX. A total of 688 YgiP-binding loci were identified after genome-wide profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites along DNA probes with a consensus GTTNATT sequence. Atomic force microscope observation indicated that at low concentrations, YgiP associates at various sites on DNA probes, but at high concentrations, YgiP covers the entire DNA surface supposedly through protein-protein contact. The intracellular concentration of YgiP is very low in growing E. coli cells under aerobic conditions, but increases more than 100-fold to the level as high as the major nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP showed retarded growth under anaerobic conditions. High abundance and large number of binding sites together indicate that YgiP is a nucleoid-associated protein with both architectural and regulatory roles as the nucleoid proteins Fis and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic conditions).


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Anaerobiose , Sequência de Bases , Sítios de Ligação , Pegada de DNA , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Genômica , Microscopia de Força Atômica , Dados de Sequência Molecular , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/genética , Fatores de Transcrição/ultraestrutura
11.
IEEE Trans Nanobioscience ; 8(4): 312-7, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19884104

RESUMO

In this paper, we describe the fabrication of a temperature-controlled microfluidic chip for cell immobilization using a thermosensitive hydrogel of poly (N-isopropylacrylamide) (PNIPAAm). A mixture of PNIPAAm solution, yeast cells, and Calcein-AM fluorescent dye is flowed in the microchannel, and the indium-tin-oxide (ITO) microheaters that were fabricated by micromachining technology heat a PNIPAAm gel. However, if the gel is very thick, it blocks the observation of the culturing cells and reduces the SNR. To address this problem, we fabricate a suspended microbridge above the microheater that limits the height of the gel, ensuring that it forms a thin and transparent layer above the heater. Microheaters and suspended biocompatible microbridge are integrated on a chip in which yeast-cell immobilization can be performed by gelation of a PNIPAAm solution.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Acrilamidas , Resinas Acrílicas , Engenharia Biomédica , Células Imobilizadas , Desenho de Equipamento , Hidrogéis , Técnicas Analíticas Microfluídicas/métodos , Polímeros , Saccharomyces cerevisiae/citologia , Temperatura
12.
Microbiology (Reading) ; 154(Pt 11): 3366-3378, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18957590

RESUMO

Purines are degraded via uric acid to yield allantoin. Under anaerobic conditions, allantoin is further degraded via carbamoylphosphate to NH(4)+ to provide a nitrogen source and, under aerobic conditions, to 3-phosphoglycerate via glyoxylate for energy production. In this study, we found that a DNA-binding transcription factor AllR, together with AllS, plays a key role in switching control of two pathways, nitrogen assimilation and energy production. The repressor function of AllR is activated in the presence of allantoin, the common substrate for both pathways, leading to repression of the genes for energy production. On the other hand, when glyoxylate is accumulated, AllR is inactivated for derepression of the pathway for energy production. RutR, the master regulator for pyrimidines and arginine, is also involved in this pathway-switching control.


Assuntos
Alantoína/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glioxilatos/metabolismo , Purinas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Sequência de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pirimidinas/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Brain Dev ; 29(8): 482-5, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17321091

RESUMO

Migraine is the most common cause of recurrent headache among children and adolescents resulting in missing of school and disabling their daily life. The purpose of this study is to determine the prevalence and clinical features of headache in junior high school children in Japan. In December 2004, questionnaires were sent to 14 junior high schools. There were multiple-choice type questions on headache, mainly migraine. The questionnaires were given during school hours, and 6472 answers were obtained. One thousand four hundred seventy-eight (22.8%) students experienced severe headache and 476 (7.4%) had consulted physicians. Three hundred thirteen (4.8%) were identified as having migraine based on the ICHD-II criteria, consisting of 110/3346 boys (3.3%) and 203/3126 girls (6.5%): 91 (29.1%) with aura and 222 (70.9%) without aura. In about half of the children the migraine attacks were of short duration, ranging from 1 to 3 h. There were 36 boys (1.1%) and 45 girls (1.4%) who had shorter attacks of less than 1 h, whom we did not diagnose as having migraine according to the ICHD-II criteria. Although migraine is common among schoolchildren, it is often under- or miss-diagnosed since the clinical figure for childhood migraine differs from that for adults.


Assuntos
Transtornos de Enxaqueca/epidemiologia , Transtornos de Enxaqueca/fisiopatologia , Adolescente , Povo Asiático , Feminino , Humanos , Japão/epidemiologia , Masculino , Transtornos de Enxaqueca/etiologia , Prevalência , Estudantes/estatística & dados numéricos , Inquéritos e Questionários
14.
FEMS Microbiol Lett ; 251(2): 243-9, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16140472

RESUMO

The ArcA/ArcB two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. In this study, uvrA gene expression was repressed when ArcA was induced in E. coli. Transcription of uvrA increased in DeltaarcA and DeltaarcB strains more than in the wild-type strain, whose trend was remarkable under the anaerobic condition. In the wild-type strain grown in the presence of DTT (10 mM), the uvrA gene expression was also repressed. Furthermore, the results of in vitro transcription and DNase I footprinting experiments indicated that ArcA specifically bound to the ArcA box [(A/T)GTTAATTA(A/T)] in the uvrA promoter and represses its transcription. These results suggest that the ArcA/ArcB two-component system works to negatively regulate uvrA gene expression.


Assuntos
Adenosina Trifosfatases/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Adenosina Trifosfatases/genética , Proteínas da Membrana Bacteriana Externa/genética , Pegada de DNA , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas Quinases/genética , Proteínas Repressoras/genética
15.
J Bacteriol ; 186(24): 8317-25, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15576781

RESUMO

In Escherichia coli K-12 overexpressing CpxR, transcription of the ung gene for uracil-DNA glycosylase was repressed, ultimately leading to the induction of mutation. Gel shift, DNase I footprinting, and in vitro transcription assays all indicated negative regulation of ung transcription by phosphorylated CpxR. Based on the accumulated results, we conclude that ung gene expression is negatively regulated by the two-component system of CpxR/CpxA signal transduction.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , DNA Glicosilases/química , DNA Glicosilases/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Proteínas Quinases/genética , Transdução de Sinais , Uracila-DNA Glicosidase
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