Biomedical subjects

Taku Oshima

Publications and source records attributed to Taku Oshima.

At least 19 recordsLinked to original sources

PubMed2006

Escherichia coli histone-like protein H-NS preferentially binds to horizontally acquired DNA in association with RNA polymerase.

Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid proteins expressed in exponentially growing Escherichia coli cells. In addition to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of gene expression. The genome-wide distribution of H-NS, compared with the distribution of RNA polymerase and transcriptionally active genes, was investigated using a high-density oligonucleotide chip. The new approach utilized in this study revealed that H-NS binds specifically to approximately 250 loci, covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase was detected in >65% of H-NS binding sites with low or no transcriptional activity, indicating that the association of RNA polymerase to promoter regions is a general mode of transcription repression by H-NS. This study also revealed that most H-NS bound DNA have been horizontally acquired, which indicates that repression of inappropriate gene expression by H-NS plays an important role in the diversification of the E. coli genome. This study presents a comprehensive assessment of the distribution of H-NS within the E. coli genome, sheds light on the mechanism underlying the transcriptional regulation by H-NS, and provides new insight into bacterial genome evolution.

Bacterial Proteins↗

PubMed2006

Large-scale identification of protein-protein interaction of Escherichia coli K-12.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.

Escherichia coli K12↗

PubMed2006

The GTP-binding protein YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis.

Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

Bacillus subtilis↗

PubMed2006

Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon.

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

Bacterial Proteins↗

PubMed2006

Identification and characterization of a second, inducible promoter of relA in Escherichia coli.

The alarmone ppGpp is an important signal molecule for the stringent response. Escherichia coli relA encodes a ppGpp synthetase, and although the regulation of RelA protein activity has been studied extensively, the regulation of relA transcription remains unclear. Here, we describe a novel relA promoter, relAP2. According to quantitative measurement of mRNA by primer extension analysis, the previously reported promoter relAP1 is constitutively active throughout growth, while relAP2 is induced temporarily at the transition state between the exponential growth and stationary phases. A chromosomal transcriptional lacZ fusion (relAP2-lacZ) showed that relAP2 is positively regulated by H-NS and CRP. Furthermore, the reduced activity of relAP2-lacZ in an hns mutant could be rescued by an rpoS mutation, which is sufficient to derepress the relAP2-lacZ activity. These data suggest that transient expression from the relAP2 promoter is controlled by several global regulators. This may account for the complex regulation of relA expression in Escherichia coli.

Base Sequence↗

PubMed2005

Cell lysis directed by sigmaE in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli.

It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding sigma(E) exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a sigma(E)-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified sigma(E) regulon genes, suggesting that a large number of genes are under the control of sigma(E) in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.

Escherichia coli↗

PubMed2004

Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli.

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

Adenosine Triphosphate↗

PubMed2004

Extracting relations between promoter sequences and their strengths from microarray data.

MOTIVATION: The relations between the promoter sequences and their strengths were extensively studied in the 1980s. Although these studies uncovered strong sequence-strength correlations, the cost of their elaborate experimental methods have been too high to be applied to a large number of promoters. On the contrary, a recent increase in the microarray data allows us to compare thousands of gene expressions with their DNA sequences. RESULTS: We studied the relations between the promoter sequences and their strengths using the Escherichia coli microarray data. We modeled those relations using a simple weight matrix, which was optimized with a novel support vector regression method. It was observed that several non-consensus bases in the '-35' and '-10' regions of promoter sequences act positively on the promoter strength and that certain consensus bases have a minor effect on the strength. We analyzed outliers for which the observed gene expressions deviate from the promoter strength predictions, and identified several genes with enhanced expressions due to multiple promoters and genes under strong regulation by transcription factors. Our method is applicable to other procaryotes for which both the promoter sequences and the microarray data are available.

Algorithms↗

PubMed2004

Analysis of hydrostatic pressure effects on transcription in Escherichia coli by DNA microarray procedure.

Hydrostatic pressure is a well-known physical stimulus, but its effects on cell physiology have not been clarified. To investigate pressure effects on Escherichia coli, we carried out DNA microarray analysis of the entire E. coli genome. The microarray results showed pleiotropic effects on gene expression. In particular, heat- and cold-stress responses were induced simultaneously by the elevated pressure. Upon temperature stress (including both temperature up- and down-shifts) and other environmental stresses, gene expression adjusts to adapt to such environmental changes through regulations by several DNA-binding proteins. An E. coli mutant, which deleted the hns gene encoding one of the regulator proteins, exhibited great pressure sensitivity. The result suggested that the H-NS protein was a possible transcriptional regulator for adaptation of the high-pressure stress.

Bacterial Proteins↗

PubMed2004

The ATPase domain of HscC (DnaK homolog) is essential for interfering sigma70 activity in E. coli.

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains. Overexpression of HscC markedly inhibits growth of host cell and reduces the sigma(70)-dependent promoter activity presumably by forming a complex with sigma(70). To identify the region(s) of HscC responsible for growth inhibition and complex formation with sigma(70), domain swapping experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the sigma(70)-dependent promoter activity and form a complex with sigma(70). These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is important for determining its functional specificity.

Adenosine Triphosphatases↗

PubMed2004

Effects of mutations in the rpoS gene on cell viability and global gene expression under nitrogen starvation in Escherichia coli.

Escherichia coli bearing an rpoS amber or disrupted mutation exhibited a significant decrease in the number of colony-forming units (c.f.u.) when exposed to nitrogen starvation, which was not observed in cells bearing a functional rpoS allele. The decrease in the number of c.f.u. that was observed about 25 h after initiation of nitrogen starvation was prevented by the addition of nitrogen within 3 h but not by the addition of nitrogen at more than 7 h after the initiation of nitrogen starvation, suggesting that a process leading to a decline in c.f.u. starts within this period. DNA microarray analysis of the rpoS mutant showed that a large number of genes including many functionally undefined genes were affected by nitrogen starvation. The expression levels of sigma(S) and sigma(H) regulon genes encoding acid-resistant proteins (hdeA, hdeB, gadA and gadB), DNA-binding protein (dps), chaperones (dnaK, ibpA, ibpB, dnaJ and htpG), chaperonins (mopB and mopA) and energy-metabolism-related proteins (hyaABCDF and gapA), and those of other genes encoding nucleotide-metabolism-related proteins (deoC and deoB), cell-division protein (ftsL), outer-membrane lipoprotein (slp) and DNA-binding protein (stpA) were significantly decreased by 10 h nitrogen starvation. The genes encoding transport/binding proteins (nac, amtB, argT, artJ, potF and hisJ) and amino acid-metabolism-related proteins (glnA, trpB, argG, asnB, argC, gdhA, cstC, ntrB, asd and lysC) were significantly up-regulated under the same condition, some of which are known Ntr genes expressed under nitrogen limitation. On the basis of these results, possible causes of the decrease in the number of c.f.u. under nitrogen starvation are discussed.

Bacterial Proteins↗

PubMed2004

Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures.

Studies of steady-state metabolic fluxes in Escherichia coli grown in nutrient-limited chemostat cultures suggest remarkable flux alterations in response to changes of growth-limiting nutrient in the medium (Hua et al., J. Bacteriol. 185:7053-7067, 2003). To elucidate the physiological adaptation of cells to the nutrient condition through the flux change and understand the molecular mechanisms underlying the change in the flux, information on gene expression is of great importance. DNA microarray analysis was performed to investigate the global transcriptional responses of steady-state cells grown in chemostat cultures with limited glucose or ammonia while other environmental conditions and the growth rate were kept constant. In slow-growing cells (specific growth rate of 0.10 h(-1)), 9.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures. One important characteristic of E. coli grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes. The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h(-1)), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations. Independent of the growth rate, 92 genes were identified as being differentially expressed. Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth.

Amino Acids↗

PubMed2004

Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems of Escherichia coli.

Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB. This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes. These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E. coli and also identified the genes that required the two systems for enhanced expression. This is one example of the intricate signal transduction networks that are posited to exist in E. coli.

Amino Acid Sequence↗

PubMed2003

Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus.

DNA microarray covering the whole genome of Staphylococcus aureus strain N315 was prepared to investigate transcription profiles. The microarray analyses revealed that vancomycin induces transcription of 139 genes. Forty-six genes among them failed to be induced in the vraSR null mutant KVR. Part of the genes regulated by VraSR system is associated with cell-wall biosynthesis, such as PBP2, SgtB and MurZ. Other cell-wall synthesis inhibitors also induced VraSR, suggesting that the sensor kinase VraS responds to the damage of cell-wall structure or inhibition of cell-wall biosynthesis. Additionally, the vraSR null mutants derived from hetero- and homo-methicillin-resistant S. aureus showed significant decrease of resistance against teicoplanin, beta-lactam, bacitracin and fosfomycin but not of D-cycloserine and levofloxacin. The observation strongly indicates that VraSR constitutes a positive regulator of cell-wall peptidoglycan synthesis, and that is deeply involved in the expression of beta-lactam and glycopeptide resistance in S. aureus.

Anti-Bacterial Agents↗

PubMed2003

Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli.

A constitutively active mutant of histidine kinase sensor EvgS was found to confer multi-drug resistance (MDR) to an acrA-deficient Escherichia coli, indicating the relationship between the two-component system EvgAS and the expression of the MDR system. The observed MDR also depended on an outer-membrane channel, TolC. Microarray and S1 mapping assays indicated that, in the presence of this constitutive mutant EvgS, the level of transcription increased for some MDR genes, including the drug efflux genes emrKY, yhiUV, acrAB, mdfA and tolC. Transcription in vitro of emrK increased by the addition of phosphorylated EvgA. Transcription activation of tolC by the activated EvgS was, however, dependent on both EvgAS and PhoPQ (Mg(2+)-responsive two-component system), in agreement with the presence of the binding site (PhoP box) for the regulator PhoP in the tolC promoter region. Transcription in vitro of yhiUV also appears to require an as-yet-unidentified additional transcriptional factor besides EvgA. Taken together we propose that the expression of the MDR system is under a complex regulatory network, including the phosphorylated EvgA serving as the master regulator.

Bacterial Outer Membrane Proteins↗

PubMed2003

Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E. coli.

Bacterial Proteins↗

PubMed2003

Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli.

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

Bacterial Capsules↗

PubMed2003

Escherichia coli response to exogenous pyrophosphate and analogs.

The addition of exogenous pyrophosphate increases the growth yield and cAMP synthesis in stationary phase when Escherichia coli is grown in minimal medium. Pyrophosphate increases the yield by altering the enterobactin uptake system. We studied the physiological effects and examined how the E. coli transcriptome was modified when two structural analogs of pyrophosphate were added to the growth medium. Methylenediphosphonic acid or a high concentration of iron had the same positive effects as pyrophosphate on growth yield, cAMP synthesis and the repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect these cellular processes significantly. The transcriptome modifications generated by pyrophosphate or methylenediphosphonic acid were more similar than those generated by imidodiphosphate or excess iron. The transcriptome data also indicated that processes other than iron uptake might be involved in the cellular response to exogenous pyrophosphate or methylenediphosphonic acid.

Culture Media↗