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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).

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.

Recoding and repurposing genetic codons By recoding bacterial genomes, it is possible to create organisms that can potentially synthesize products not commonly found in nature. By systematic replacement of seven codons with synonymous alternatives for all protein-coding genes, Ostrov et al. recoded the Escherichia coli genome. The number of codons in the E. coli genetic code was reduced from 64 to 57 by removing instances of the UAG stop codon and excising two arginine codons, two leucine codons, and two serine codons. Over 90% functionality was successfully retained. In 10 cases, reconstructed bacteria were not viable, but these few failures offered interesting insights into genome-design challenges and what is needed for a viable genome. Science , this issue p. 819

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.

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.

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.

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).

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.

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.

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.

Transcription of the Escherichia coli genome is under the control of approximately 300 species of transcription factor. However, the regulatory roles remain unidentified for about 100 species of the transcription factor. As an approach to identify their regulation modes, we analyzed the dynamics of DNA-transcription factor interactions using the atomic force microscopy (AFM). Here we examined the DNA-binding mode of two types of transcription factor in the presence and absence of RNA polymerase: a novel growth-specific nucleoid protein Dan (DNA-binding protein under anaerobic growth conditions); and global regulators, CRP (cAMP receptor protein) and Cra (catabolite repressor and activator), for control of transport and metabolism of carbon sources.

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.

Thiacrown ether additive enhanced enantioselectivity in lipase-catalyzed trans-esterification of allyl alcohols. Small amounts of thiacrown ether offered highly enantioselective reaction when 5-phenylpentene-3-ol was subjected to the reaction of Pseudomonas cepacia lipase.

For measurement of the promoter activity and regulation within single bacterial cells, we constructed various types of the cell chip. After gelation of portions of E. coli culture wihin the cell chip, we have succeeded, for the first time, the real-time single-bacterial cell assay of promoter activity. Here we applied this newly developed system for measurement of the activity and regulation of E. coli gcl promoter.

A systematic search has been performed for DNA-binding sequences recognized by YgiP, an uncharacterized transcription factor of Escherichia coli, using the newly developed Genomic SELEX. By SELEX-clos procedure, a total of 333 sequences have been isolated from a total of 126 loci of the E. coli genome while more than 703 YgiP-binding loci have been identified after the genome-wide profiling by SELEX-chip procedure. Gel mobility shift assay indicated the presence of multiple YgiP-binding sites along each SELEX DNA fragment while DNase-I foot-printing assay indicated protection of short-specific sequences by YgiP, together suggesting YgiP as a nucleoid protein with broad specificity of DNA binding. Atomic force microscope (AFM) observation indicated that at low concentrations, YgiP binds to various sites in non-specific manner, but at high concentrations, YgiP covers the entire DNA surface. The intracellular level of YgiP was found to be very low in steady-state of cell growth under aerobic conditions, but increased more than 100-fold to the level as high as those of HU and IHF under hypoxic or anaerobic culture conditions. An E. coli mutant lacking ygiP showed abnormal growth under anaerobic conditions. Taken together we conclude that YgiP is a novel nucleoid protein of E. coli under the anaerobic conditions, and thus propose to rename YgiP to Dan (DNA-binding protein under anaerobic conditions).

For measurement of the promoter activity and regulation within single bacterial cells, we constructed various types of the cell chip. After gelation of portions of E. coli culture using the cell chip, we have succeeded, for the first time, the real-time single-bacterial cell assay of E. coli gcl promoter strength.

This paper presents a temperature sensing and controlling microfluid chip for cell immobilization using a thermo-sensitive hydrogel (PNIPAAm). The ITO (Indium Tin Oxide) microheaters fabricated by micromachining technology, perform heating of the solution of cell, PNIPAAm and Calcein-AM (fluorescent dye) in microchannels. It is important to note that a thick gel formation cause to block the observation of culturing cells due to the autofluorescence of gel which reduce the S/N ratio. For the present study, we are targeting to fabricate a suspended micro-bridge above a microheater to limit the height of gel to form "thin and transparent gel layer" above the heater. The research was focused on controlling of gel thickness which eliminated unnecessary interferences of autofluorescence of gel in the observation by the inverted microscope, and a better S/N ratio can be obtained than the conventional observation with uncontrolled gel thickness. All the heating and a suspended biocompatible micro-bridge were integrated on a chip, in which yeast cells immobilization can be performed by the gelation of the PNIPAAm solution.

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.

The modulation of gene selectivity of RNA polymerase is the key step in the bacterial response to environmental changes. The gene selectivity of RNA polymerase is modulated after interaction with seven species of sigma factor, the promoter recognition subunit, and a total of about 300 DNA-binding transcription factors in the second step. In order to get insights into the molecular mechanisms underlying the global regulation of genome expression, our effort has been focused on the identification of genes and promoters under the control of each transcription factor. For this purpose, we have developed the 'Genomic SELEX' system, which allows the quick identification of DNA sequences recognized in vitro by transcription factors. Regulation in vivo of the target genes predicted after the 'Genomic SELEX' was examined by analysis of individual transcripts in the presence and absence of the test transcription factors. The regulated promoter activity was also examined using the newly constructed two-fluorescent-protein promoter assay vector pGRP. Results altogether indicate the involvement of complex networks between transcription factors and promoters in the global regulation of genome transcription.

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.

The pattern of genome transcription in Escherichia coli determined by controlling the distribution of a total of about 2,000 molecules of RNA polymerase, the transcriptase, among 4,000 genes on its genome. In response to environmental changes, the gene selectivity of the RNA polymerase is modulated after interaction with one sigma subunit, the promoter recognition subunit, in the first step and one or two of about 300 transcription factors in the second step. In order to get insights into the molecular mechanisms underlying the global regulation of genome expression, we have initiated a systematic study of the identification of genes under the control of each transcription factor using the newly developed 'Genomic SELEX' system. The gene regulation predicted by the Genomic SELEX is being examined by analysis of the promoter activity in the presence and absence of test transcription factors using the newly constructed two-fluorescent-protein promoter assay vector pGRP. Results altogether indicate the involvement of complex networks between transcription factors and promoters in the global regulation of genome transcription

The model bacterium, Escherichia coli, is able to survive under various stressful conditions in nature. From the complete genome sequence, about 4,400 genes are considered to exist on the genome. Selective and regulated expression of the genes is critical for bacterial adaptation to environment. Since the number of RNA polymerase, the enzyme responsible for transcription, is less than that of total genes, we proposed that the controlled distribution of RNA polymerase among 4,400 genes determines the genome expression pattern. In order to understand the molecular basis of genome regulation, we have been concerned with the analysis of all 300 transcription factors, which are involved in the functional modulation of RNA polymerase for switching the gene selectivity. For this purpose, we have performed the systematic search for target genes and promoters recognized by each transcription factor using the newly developed genomic SELEX system. Here we describe some results of this Multi-scale Genetics focusing on the protein-protein and protein-DNA interaction networks involved in the genome regulation. One hitherto undescribed finding is that the transcription factors form interplay networks, in which one transcription factor controls the expression of another factor and so on. The putative transcription factors with unidentified functions are often located on top of the hierarchy of transcription factor cascade. Such networks appear to be needed for stress response in nature.

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.

Cerebral ischemia has been proposed as the etiology of transient global amnesia. Recently, however, migranous and epileptic etiologies have attracted attention. A 56-year-old man had transient global amnesia and the next day began to display symptoms of meningoencephalitis. Herpes simplex encephalitis was diagnosed based on the titer of herpes simplex virus antibodies. The transient global amnesia appears to have occurred as an early sign of herpes simplex encephalitis and may have been provoked by an epileptic mechanism.

We report a 52-year-old man with idiopathic thrombocytopenic purpura accompanying acute oculomotor nerve palsy. At autopsy, intraneural hemorrhage was found in the cavernous portion of the oculomotor nerve. Intraneural bleeding as well as extraneural compression by hematoma formation should be considered as a cause of neuropathies occurring in patients with aggravated thrombocytopenia.

Thiacrown ether additive enhanced enantioselectivity in lipase-catalyzed trans-esterification of allyl alcohols. Small amounts of thiacrown ether offered highly enantioselective reaction when 5-phenylpentene-3-ol was subjected to the reaction of Pseudomonas cepacia lipase.

29才,男性. 27才の時に肩甲部痛,背部・腰部痛とともに舌の右への偏位に気付いた.その後,胸部絞扼感,乾性咳嗽,下肢脱力感が出現したため,入院.胸部X線写真で多量の胸水と左肺に円形陰影を認めた.胸部CTで左後部胸腔内の腫瘤と判明した.胸水細胞診にて異型形質細胞を証明した.血清M成分は認めず血中IgA, IgG, IgM, IgE, IgDは全て低値であつたが,尿中にはκ型のBence Jones蛋白を認めた.髄液蛋白の著明増加も認めた.舌は右へ偏位し線維束攣縮を認めた.入院後数日にしてTh5以下の横断性脊髄障害を来した.入院2カ月後には項部硬直も出現し,腫瘤形成性形質細胞腫による横断性脊髄障害と髄膜癌腫症と考え,放射線治療,薬物治療を行なつたが,全経過14カ月で死亡した.剖検では肉眼的には脳硬膜・軟膜には肥厚や混濁を認めず,頭蓋底部にも異常を認めなかつた.脊髄はTh5のレベルで腫瘤により圧迫されており,その部分より下約10cmにわたつて脊髄は軟らかく細くなつていた.光顕的には髄膜癌腫症と腫瘤の脊髄への圧迫壊死が判明した.舌下神経麻痺を初発とする髄膜癌腫症はまれであり,また過去に腫瘤形成性形質細胞腫による髄膜癌腫症と横断性脊髄症状の報告はない.

門脈から右腎静脈を介し,下大静脈へ流入する側副血行路を伴う肝外門脈閉塞型の門脈圧亢進症の症例を報告した.患者は50歳女性で,手指振戦,歩行障害を呈し,入院した.さらに,脾腫,汎血球減少,高アンモニア血症が認められ,脳波では,三相波が認められた.腹腔動脈造影,逆行性腎静脈造影により,本例を右腎静脈門脈側副血行路を伴う肝外門脈閉塞型の門脈圧亢進症であると診断した.脾機能亢進が疑われ,食道静脈瘤が認められなかったので,脾摘術を施行した.術後,末梢血検査成績は改善した.また,食餌性蛋白の制限,ラクチュロースの経口投与により,高アンモニア血症は正常化し,神経症状も徐々に改善した.本例の肝性脳症は巨大な後腹膜性の側副血行路に原因したと思われる.本例は右腎静脈を介し,下大静脈へ流入する門脈系側副血行路を診断し得た点で,稀有な症例であった.

症例は45才の男性.糖尿病の治療経過中,両下肢に多発性神経炎を伴い肢端紅痛症が出現した.症状は約半年間にわたり増悪し, aspirinその他の鎮痛薬や血管拡張剤は無効であった. Mecobalaminの静注を試みたところ開始数日後より肢端紅痛症状は著明な改善傾向を示し,引き続き多発性神経炎も軽減した.臨床経過および薬効より,末梢神経障害が肢端紅痛症状の発現に関与したと推察された.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of promoterless genes embedded in cassettes. Some integrons, like the one found in the second chromosome ofVibrio cholerae, can be particularly massive, gathering hundreds of non-expressed cassettes. It is unclear how such genetic structures can be stabilized in bacterial genomes. Here, we reveal that the orientation of integrons toward replication within bacterial chromosomes is essential to their stability. Indeed, we show that upon inversion of theV. choleraechromosomal integron, the cassette excision rate of its cassettes is dramatically increased. This correlates with a strong growth defect which we show is mostly due to the excision of a remarkable type of cassettes bearing their own promoter and encoding toxin– antitoxin systems. This “abortive excision” of toxin–antitoxin systems can prevent the inversion of chromosomal integrons and the associated extensive loss of cassettes. Our analysis of the available sedentary chromosomal integrons in genome database show an over-representation of toxin–antitoxin cassettes in large integrons. This study thus provides a striking example of the relationship between genome organization, genome stability and an emerging property of toxin–antitoxin systems.