Abstract: Cellobiose triggers the production of two endoglucanases, EglA and EglB, in Aspergillus nidulans. The cellulose responsive element (CeRE) cis-element that is essential for induction has been identified on the eglA promoter, but transcription factors that bind to CeRE have not yet been identified. CeRE contained a consensus sequence CC(A/T)(6)GG for binding of the SRF-type MADS box proteins. Introduction of a missense mutation into mcmA, encoding for the sole SRF-MADS protein in A. nidulans, caused a significant reduction in cellulase induction. Real-time RT-PCR analysis revealed that inductive expression of not only eglA but also eglB and cbhA by cellobiose were under control of McmA. The McmA protein expressed in Escherichia coli specifically bound to two regions of the eglA promoter: CeRE and its upstream proximal region. These results, together with our previous study on the eglA promoter structure, imply that McmA regulates eglA expression by binding directly to its promoter. This is the first evidence for participation of an SRF-MADS protein in cellulase regulation.
Abstract: AmyR, a fungal transcriptional activator responsible for induction of amylolytic genes in Aspergillus nidulans, localizes to the nucleus in response to the physiological inducer isomaltose. Maltose, kojibiose, and D: -glucose were also found to trigger the nuclear localization of GFP-AmyR. Isomaltose- and kojibiose-triggered nuclear localization was not inhibited by the glucosidase inhibitor, castanospermine, while maltose-triggered localization was inhibited. Thus, maltose itself does not appear to be an direct inducer, but its degraded or transglycosylated product does. Non-metabolizable D: -glucose analogues were also able to trigger the nuclear localization, implying that these sugars, except maltose, directly function as the inducers of AmyR nuclear entry. The inducing activity of D: -glucose was 4 orders-of-magnitude weaker compared with isomaltose. Although D: -glucose has the ability to induce α-amylase production, this activity would generally be masked by CreA-dependent carbon catabolite repression. Significant induction of α-amylase by D: -glucose was observed in creA-defective A. nidulans.
Abstract: XlnR is a transcription factor that mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. In order to clarify the molecular mechanisms underlying XlnR-mediated induction, Aspergillus oryzae XlnR was fused with the c-myc tag and examined by Western blotting. Phosphate-affinity SDS-PAGE revealed that XlnR was present as a mixture of variously phosphorylated forms in the absence of D-xylose, and that D-xylose triggered additional phosphorylation of the protein. D-Xylose-triggered phosphorylation was a rapid process occurring within 5 min prior to the accumulation of xynG2 mRNA, and removal of D-xylose caused slow dephosphorylation, leading to less-phosphorylated forms. At 30 min after removal, the phosphorylation status was almost identical to that in the absence of D-xylose, and the level of xynG2 mRNA started to decrease. These results indicate that XlnR is highly phosphorylated when it is active in transactivation, implying that D-xylose-triggered reversible phosphorylation controls XlnR activity.
Abstract: In this study, saccharification of the inner bark of Eucalyptus was carried out by enzymatic hydrolysis to produce bioethanol from non-food biomass. To enhance the accessibility of the enzyme to the polysaccharides such as cellulose and holocellulose in the cell wall of the bark, the bark was subjected to hydrothermal pre-treatment with carbon dioxide. This pre-treatment considerably influenced enzymatic hydrolysis. The main component (over 90%) of the generated monosaccharide was glucose, and the yield of glucose on the basis of alpha-cellulose reaches about 80%. This result suggests that the secondary wall, whose main component is cellulose, was effectively hydrolyzed by the enzyme. Microscopic analysis revealed that after pre-treatment, the phloem parenchyma cell had a considerably swollen primary wall and the phloem fibre showed many nano-clefts within its secondary wall. These structural changes appeared to promote enzymatic hydrolysis, because of high accessibility of enzymes to cellulose in the secondary wall.
Abstract: We established a high-throughput screening method for the DNA binding sequence of a eukaryotic transcription factor by using the bead display system with emulsion PCR and flow cytometry and applied it for identifying a eukaryotic transcriptional activator AmyR, which is known to regulate amylolytic gene expression in Aspergillus species. Segmented parts of the binding site of AmyR were randomized to make a DNA library on beads, onto which MalE-tagged AmyR protein and fluorescent anti-MalE tag antibody were bound, followed by selection with a flow cytometer. From a library replacing well-conserved six nucleotides (CGG-CGG) to random ones, the consensus sequence was recovered at a high frequency, demonstrating the reliability of the screening system. Interestingly, similar analysis for another library having randomized intermediate eight nucleotides between the conserved triplets revealed that the selected intermediate sequence had a strong preference for the T nucleotide. Moreover, exactly the same sequence with the upstream region of amyB, a typical AmyR-regulated gene, was found in the selection. These results suggest that this screening system will be a powerful tool for high-throughput analysis of the eukaryotic transcriptome.
Abstract: XlnR is a Zn(II)2Cys6 transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus. Overexpression of the aoxlnR gene in Aspergillus oryzae (A. oryzae xlnR gene) resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were detected by two-dimensional electrophoresis. DNA microarray analysis to identify the transcriptional targets of AoXlnR led to the identification of 75 genes that showed more than fivefold increase in their expression in the AoXlnR overproducer than in the disruptant. Of these, 32 genes were predicted to encode a glycoside hydrolase, highlighting the biotechnological importance of AoXlnR in biomass degradation. The 75 genes included the genes previously identified as AoXlnR targets (xynF1, xynF3, xynG2, xylA, celA, celB, celC, and celD). Thirty-six genes were predicted to be extracellular, which was consistent with the number of proteins secreted, and 61 genes possessed putative XlnR-binding sites (5'-GGCTAA-3', 5'-GGCTAG-3', and 5'-GGCTGA-3') in their promoter regions. Functional annotation of the genes revealed that AoXlnR regulated the expression of hydrolytic genes for degradation of beta-1,4-xylan, arabinoxylan, cellulose, and xyloglucan and of catabolic genes for the conversion of D-xylose to xylulose-5-phosphate. In addition, genes encoding glucose-6-phosphate 1-dehydrogenase and L-arabinitol-4- dehydrogenase involved in D-glucose and L-arabinose catabolism also appeared to be targets of AoXlnR.
Abstract: AmyR is a Zn(II)(2)Cys(6) transcriptional activator that regulates expression of the amylolytic genes in Aspergillus species. Subcellular localization studies of GFP-fused AmyR in A. nidulans revealed that the fusion protein preferentially localized to the nucleus in response to isomaltose, the physiological inducer of the amylolytic genes. The C-terminal domains of AmyR, designated MH3 (residues 419-496) and MH4 (residues 516-542), were essential for sensing the inducing stimulus and regulating the subcellular localization. The MH2 domain (residues 234-375) located in the middle of AmyR was required for transcriptional activation of the target genes, and the nuclear localization signals were identified within the N-terminal Zn(II)(2)Cys(6) DNA binding motif.
Abstract: The transcriptional activator XYR1 is the central regulator that governs cellulolytic and xylanolytic gene expression in Trichoderma reesei. However, despite its biological importance, relatively little is known about its functional binding sequences. In the present study, we investigated the binding characteristics and specific target for XYR1 by using DNase I footprinting analysis and electrophoretic mobility shift assays. We demonstrate that XYR1 can interact not only with the 5'-GGCTAA-3' motif but also with several 5'-GGC(A/T)(3)-3' motifs. In silico analysis revealed that the 5'-GGC(A/T)(3)-3' motifs are widespread as single site in 5'-upstream region of all the XYR1-regulated genes. Furthermore, we defined the important nucleotides within the binding site that contribute to specific interaction with XYR1. Our results suggest that, together with the inverted repeat motifs, the single 5'-GGC(A/T)(4)-3' motifs play important roles as functional XYR1-binding sites in the regulation of cellulase and xylanase gene expression in T. reesei.
Abstract: The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
Abstract: Expression of the eglA gene, encoding for a major endoglucanse EG A in Aspergillus nidulans, is induced by cellulose and cellobiose, but not by xylose. This suggests that induction is independent of XlnR, a transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus.Mutational analysis of the eglA promoter was performed to identify the novel cis-element responsible for XlnR-independent induction. The region spanning -153 to -138 (CCGTACCTTTTTAGGA), designated CeRE(Cellulose Responsive Element), was found to be the upstream activating element essential to inductive expression of the eglA gene.
Abstract: The xylanase III gene (xyn3) from the filamentous fungus Trichoderma reesei PC-3-7 is only induced by cellulose, its derivatives and L-sorbose, but not by xylan. In this study, we defined three cis-acting elements within the xyn3 upstream region by using detailed deletion and mutation analysis. In addition to the Xyr1/ACEII-binding motif (5'-GGCTAA-3'), the analogous motifs 5'-GGCTAT-3' and 5'-GGCAAA-3' presented as an inverted repeat spaced by 16-bp internal sequences were identified as essential elements for xyn3 expression. Electrophoretic mobility shift assay using heterologously expressed Xyr1 demonstrates that all the identified cis-acting elements are able to interact with Xyr1. Furthermore, no xyn3 transcripts were formed in the xyr1-knockout strain upon induction by sophorose and L-sorbose. These results indicate that xyn3 expression is transcriptionally regulated by Xyr1, and suggest that the 5'-GGCTAT-3' and 5'-GGCAAA-3' motifs play roles in Xyr1-mediated cellulase and xylanase gene expression in T. reesei.
Abstract: His-Asp phosphorelays are signal transduction mechanisms widely found in both prokaryotes and eukaryotes. The phosphorelay comprises three types of signal transducers: a sensor with histidine kinase (HK), a response regulator containing a phospho-accepting receiver (RR), and a histidine-containing phosphotransmitter (HPt). In this study, we examined HK expression using a green fluorescent protein (GFP) reporter driven by HK promoters in Aspergillus nidulans. All the transformants showed fluorescence derived from GFP in a submerged culture, although some of them were very weak, indicating that all the promoters were active. As judged by the fluorescence of transformants grown on a culture plate on which sexual development was induced, promoters of fphA, hk-8-2, and hk-8-5 preferentially functioned in conidial heads, the promoter of phkA preferentially functioned in cleistothecia, and the promoters of tcsB and nikA function in both conidial heads and cleistothecia. These results indicate that at least some HKs of A. nidulans showed temporally and spatially different expression during the cell cycle.
Abstract: In the filamentous fungus Trichoderma reesei, endoglucanase III (EGIII) is coordinately expressed with other cellulases during growth on cellulose, its derivatives, and L-sorbose. To elucidate EGIII induction mechanism, we cloned and sequenced the upstream region of egl3 encoding EGIII. Two GGCTAA motifs, a putative binding site for ACEII and xylanase regulator Xyr1, were found on the template strand of the egl3 upstream region. Deletion analysis of the egl3 upstream region using the beta-glucuronidase (GUS) reporter system revealed that removal of regions containing the GGCTAA motifs and the region between -1,045 and -1,002 bp containing GGCTAT motif severely affected GUS inducibility. Furthermore, mutation of the two GGCTAA motifs and the GGCTAT motif of this region led to a significant decrease in GUS activity. These data indicate that both GGCTAA and GGCTAT are key motifs for egl3 expression, and that egl3 induction may also be controlled by Xyr1. This hypothesis was supported by in vitro electrophoretic mobility shift assay, in which heterologously expressed Xyr1 specifically bound not only GGCTAA but also GGCTAT motif.
Abstract: His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study.
Abstract: Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.
Abstract: We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.
Abstract: The genome sequence of Aspergillus oryzae, a fungus used in the production of the traditional Japanese fermentation foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste), has revealed prominent features in its gene composition as compared to those of Saccharomyces cerevisiae and Neurospora crassa. The A. oryzae genome is extremely enriched with genes involved in biomass degradation, primary and secondary metabolism, transcriptional regulation, and cell signaling. Even compared to the related species A. nidulans and A. fumigatus, an abundance of metabolic genes is apparent, with acquisition of more than 6 Mb of sequence in the A. oryzae lineage, interspersed throughout the A. oryzae genome. Besides the various already established merits of A. oryzae for industrial uses, the genome sequence and the abundance of metabolic genes should significantly accelerate the biotechnological use of A. oryzae in industry.
Abstract: Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.
Abstract: The CCAAT-binding complex in Aspergillus species, known as the Hap complex, consists of at least three subunits, HapB, HapC, and HapE. Each Hap subunit contains an evolutionarily conserved core domain. In this study, a series of the truncated gene, which encodes the HapE subunit of Aspergillus oryzae, was constructed to survey the regions essential for the transcriptional enhancement of fungal genes. It was revealed that the non-conserved regions and the conserved region similar to the Hap4p recruiting domain of Saccharomyces cerevisiae were not necessary for Hap complex-mediated transcriptional enhancement.
Abstract: Aspergillus nidulans possessed 16 putative amylolytic genes consisting of 7 alpha-glucosidase (agdA-F), 7 alpha-amylase (amyA-F), and 2 glucoamylase (glaA and B) genes on the genome. Among them, the agdA, agdB, agdE, agdF, amyA, amyB, amyF, and glaB genes were induced by isomaltose. AmyR, a Zn(II)(2)Cys(6) transcription factor, was required for the induction. The isomaltose-inducible genes possessed at least one consensus sequence for AmyR binding, 5'-CGGN(8)CGG, on each promoter region. None of the amylolytic genes was induced by maltose. The mRNA levels of the amylolytic genes except for agdC, amyD, and amyG increased under carbon-starved conditions. Release from CreA-dependent carbon catabolite repression was the main cause of the increase, but, the mRNA levels of agdB, agdF, amyB, amyF, and glaB increased to some extent even in a creA mutant. Therefore, both CreA-dependent and -independent mechanisms are involved in the up-regulation of the amylolytic genes under carbon-starved conditions.
Abstract: The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a 'piggy back mechanism' in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.
Abstract: Filamentous fungi are frequently used for the production of industrial enzymes, since they produce a variety of enzymes including polysaccharide-degrading enzymes. Among the many filamentous fungi, Aspergillus species, such as A. oryzae and A. niger, are known as strong producers of amylolytic enzymes. We have been studying on the regulatory mechanisms underlying the expression of A. oryzae amylolytic genes. Based on analyses using a hybrid model system of A. nidulans transformed by a gene encoding A. oryzae Taka-amylase A, the major amylase (taaG2), we have found that three factors, CCAAT-box binding protein, CreA, and AmyR, are involved in taaG2 gene expression and regulation. In this review, the focus is on the CCAAT-box binding protein of filamentous fungi. The assembly, nuclear translocation, and transcriptional enhancement mechanisms of the CCAAT-box binding protein are discussed.
Abstract: The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.
Abstract: AmyR is a transcriptional activator in Aspergillus spp. necessary for induction of the amylolytic enzyme genes. It recognizes 5'-CGGN8CGG-3' conserved in a number of the amylolytic gene promoters, and in addition 5'-CGGAAATTTAA-3' in the A. oryzae alpha-amylase promoter. In this report, interaction of AmyR with the 5'-CGGAAATTTAA-3' type binding site in the Taka-amylase gene (taaG2) promoter was precisely characterized by DNase I footprinting analysis and electrophoretic mobility shift assay in vitro, and also by examination of the in vivo activity of the mutated promoters. The in vitro and in vivo analyses indicated that two AmyR molecules bind cooperatively to the 5'-CGGAAATTTAA-3' sequence by recognizing the CGG triplet at the 5'-end and the AGG triplet just downstream of the sequence.
Abstract: An unresolved question concerns the nuclear localization of the heterotrimeric CCAAT-binding complex, which is evolutionarily conserved in eukaryotic organisms including fungi, plants and mammals. All three subunits are necessary for DNA binding. In the filamentous fungus Aspergillus nidulans the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the HapB, HapC and HapE subunits. Here, by using various green fluorescent protein constructs, a nuclear localization signal sequence (NLS) of the HapB protein was identified, outside of the evolutionarily conserved domain. HapB-EGFP was transported into the nucleus in both DeltahapC and DeltahapE strains, indicating that its NLS interacts with the import machinery independently of the other Hap subunits. In contrast, HapC-EGFP did not enter the nucleus in the absence of HapE or HapB. A similar finding was made for HapE-EGFP, which did not localize to the nucleus in the absence of HapC or HapB. Addition of the HapB-NLS to either HapC or HapE led to nuclear localization of the respective protein fusions, indicating that both HapC and HapE lack a functional NLS. Furthermore, these data strongly suggest that HapC and HapE have first to form a heterodimer and can be transported only as a heterodimer via the HapB protein into the nucleus. Therefore, the HapB subunit is the primary cargo for the import machinery, while HapC and HapE are transported to the nucleus only as a heterodimer and in complex with HapB via a piggy back mechanism. This enables the cell to provide equimolar concentrations of all subunits to the nucleus.
Abstract: In Aspergillus oryzae, inductive expression of the xynF1 gene is mediated by a transcriptional activator, AoXlnR. Promoter activity of the xynF1 gene, monitored by beta-galactosidase activity, was successfully upregulated by mutating two non-canonical AoXlnR binding sequences to what is thought to be the strongest sequence. Transformants carrying three canonical binding sequences in the promoter region produced enzyme 2.8 times more active (33,000 units mg(-1) protein) than that of transformants carrying the authentic promoter (12,000 units mg(-1) protein).
Abstract: AIM: In vivo regulatory features of AnRP, a repressor for the cgxA gene, were characterized. METHODS AND RESULTS: Titration of AnRP by introducing multiple copies of its specific binding sequence 'TTGACAAAT' into an Aspergillus nidulans strain containing the Chaetomium gracile cgxA gene enhanced the cgxA gene expression. AnRP functions independently of and cooperatively with CreA to modulate the cgxA gene expression. CONCLUSIONS: AnRP is a CreA-independent negative regulatory factor controlling the cgxA expression. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Xylanases of filamentous fungi have received increased interest because of their potential biotechnological applications. Elucidation of the factors involved in the regulation of the xylanolytic genes in fungi will help to increase levels of xylanase production.
Abstract: Among various alpha-glucobioses examined, isomaltose was the most effective inducer for amylase synthesis in Aspergillus nidulans. Amylase induction by maltose was completely inhibited by addition of castanospermine or cycloheximide, while induction by isomaltose was not affected by the inhibitors, suggesting that amylase induction by maltose requires inducible alpha-glucosidases. Disruption of the alpha-glucosidase A gene ( agdA), the alpha-glucosidase B gene ( agdB), or both genes did not abolish maltose-dependent induction, although amylase production induced by maltose decreased about 2-fold in the agdA/ agdB double disruptant, compared with that in the agdB disruptant at all concentrations tested. Upon induction by isomaltose, amylase synthesis was enhanced considerably in the agdB and agdA/ agdB disruptants. Even at 3 nM, isomaltose induced amylase production in the double disruptant, supporting the suggestion that isomaltose is a physiological inducer for amylase. Therefore, maltose must be converted to isomaltose by alpha-glucosidases prior to triggering amylase synthesis, but no specific alpha-glucosidase is required for amylase induction by maltose. Probably any alpha-glucosidases having isomaltose-forming activity, including AgdA and AgdB, may participate in amylase induction by maltose.
Abstract: A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55-56%).
Abstract: AoXlnR was isolated as a transcriptional activator of the major xylanase gene, xynF1, in Aspergillus oryzae. To investigate the spectrum of genes under the control of AoXlnR, expression of the xylanolytic and cellulolytic genes in an A. oryzae wild type strain, an AoxlnR disruptant and an AoXlnR overexpressed strain was analyzed by Northern blotting. AoXlnR mediated expression of at least four xylanolytic genes and four cellulolytic genes when induced by xylan and D-xylose. Moreover, AoXlnR was newly found to mediate the cellulose-inductive expression of the xylanolytic genes as well as the cellulolytic genes.
Abstract: The Saccharomyces cerevisiae CCAAT-binding factor is composed of four subunits Hap2p, Hap3p, Hap4p and Hap5p. Three subunits, Hap2/3/5p, are required for DNA-binding and Hap4p is involved in transcriptional activation. Although homologues of Hap2/3/5p (in the case of Aspergillus nidulans; HapB/C/E, respectively) were found in many eukaryotes, no Hap4p homologues have been found except for the other yeast, Kluyveromyces lactis. With the lexA-hap2, -hapB, -hapC, or -hapE fusion gene, we evaluated the ability of interaction between Aspergillus Hap subunits and S. cerevisiae Hap4p subunit in S. cerevisiae. Using the system with lexA-hapB, a gene encoding a novel transcriptional activator, which interacted with the Hap complex, was isolated from A. nidulans and designated hapX.
Abstract: The Aspergillus nidulans CCAAT-binding complex (Hap complex) consists of at least three subunits, HapB, HapC and HapE. To investigate the quantity control mechanisms of the subunits during assembly of the Hap complex, reconstitution studies with the recombinant subunits and extracts prepared from the respective hap subunit deletion mutants were carried out. Furthermore, Western blot analysis of the Hap subunits and Northern blot analysis of the hap genes with the respective deletion mutants were also performed. From all the results together, it was suggested that the number of the HapC molecule could adjust that of the HapE molecule by forming stable heterodimers prior to assembly of the Hap complex.
Abstract: Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.
Abstract: A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5-7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.
Abstract: By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.
Abstract: Two genes, AohapB and AohapE, encoding subunits of the Aspergillus oryzae CCAAT-binding complex were cloned and sequenced. The polypeptides encoded by AohapB and AohapE were expressed in Escherichia coli and used to reconstitute a DNA-binding complex with recombinant AoHapC. The DNA-binding activity was observed only in the presence of all three subunits, indicating that AoHapB, AoHapE and AoHapC are essential for CCAAT-binding. Furthermore, introduction of the AohapB, AohapC and AohapE genes into the A. nidulans hapB delta, hapC delta and hapE delta strains, respectively, revealed that the A. oryzae Hap subunits are functionally interchangeable with the corresponding subunits in A. nidulans.
Abstract: To examine the possibility that factors different from the Hap complex are involved in increasing Taka-amylase A gene (taa) expression in Aspergillus nidulans, either the authentic taa gene or the mutant taa gene with a replacement of the CCAAT box was expressed in either a wild type strain or a mutant strain lacking the functional Hap complex (hapCdelta). When the mutant taa was expressed in the hapCdelta strain, enzyme activity was as low as that of the hapCdelta strain transformed with the authentic taa gene, indicating that no factors except for the Hap complex increase transcription of the taa gene by binding to the CCAAT sequence.
Abstract: The alpha-glucosidase gene (agdA) of Aspergillus nidulans has a single CGGN8CGG type AmyR binding site in its promoter region. The binding site is functional in vivo as a cis-element responsible for induction by starch, and mutational studies indicated that both the CGG triplets are required for high-level induction. A part of AmyR (residues 1-411; AmyR(1-411)), which was produced as a MalE fusion protein in E. coli, bound to the CGGN8CGG site of the agdA promoter. DNA binding profiles to the mutant binding sites that lacked both or either one of the CGG triplets suggested that AmyR(1-411) can bind to a single CGG triplet site with low affinity and that two AmyR molecules cooperatively bind to the CGG direct repeat.
Abstract: Filamentous fungi produce high levels of polysaccharide-degrading enzymes and are frequently used for the production of industrial enzymes. Because of the high secretory capacity for enzymes, filamentous fungi are effective hosts for the production of foreign proteins. Genetic studies with Aspergillus nidulans have shown pathway-specific regulatory systems that control a set of genes that must be expressed to catabolize particular substrates. Besides the pathway-specific regulation, wide domain regulatory systems exist that affect a great many individual genes in different pathways. A molecular analysis of various regulated systems has confirmed the formal models derived from purely genetic data. In general, many genes are subject to more than one regulatory system. In this article, we describe two transcriptional activators, AmyR and XlnR, and an enhancer, Hap complex, in view of their regulatory roles in the expression of the amylolytic and (hemi-)cellulolytic genes mainly in aspergilli. The amyR gene has been isolated as a transcriptional activator involved in the expression of amylolytic genes from A. oryzae, A. niger, and A. nidulans, and the xlnR gene, which has been isolated from A. niger and A. oryzae, activates the expression of xylanolytic genes as well as some cellulolytic genes in aspergilli. Both AmyR and XlnR have a typical zinc binuclear cluster DNA-binding domain at their N-terminal regions. Hap complex, a CCAAT-binding complex, enhances the overall promoter activity and increases the expression levels of many fungal genes, including the Taka-amylase A gene. Hap complex comprises three subunits, HapB, HapC, and HapE, in A. nidulans and A. oryzae as well as higher eukaryotes, whereas HAP complex in Saccharomyces cerevisiae and Kluyveromyces lactis has the additional subunit, Hap4p, which is responsible for the transcriptional activation. Hap complex is suggested to enhance transcription by remodeling the chromatin structure. The regulation of gene expression in filamentous fungi of industrial interest could follow basically the same general principles as those discovered in A. nidulans. The knowledge of regulation of gene expression in combination with traditional genetic techniques is expected to be increasingly utilized for strain breeding. Furthermore, this knowledge provides a basis for the rational application of transcriptional regulators for biotechnological processes in filamentous fungi.
Abstract: The Aspergillus nidulans amyR gene and its cDNA were cloned and sequenced. The genomic gene comprised 2,092 bp, interrupted by two short introns, and encoded a cys-6 zinc transcriptional activator (AMYR) of 662 amino acid residues with a calculated molecular mass of 72,862 Da. Disruption of the amyR gene caused defects in the utilization of maltose and starch and abolished expression of the taaG2 gene encoding A. oryzae Taka-amylase A, which is inducibly and abundantly expressed in the wild-type A. nidulans. Expression of the amyR gene was under the control of the carbon catabolite repressor, CREA. The growth defect of the malA1 mutant on maltose was complemented by the amyR gene; and the amyR gene derived from the mutant possessed a single mutation, from A to T, at position 1,483, resulting in a substitution of His478 to Leu. These results indicate that the amyR gene is identical to the genetically defined malA gene. AMYR possessed five domains (Zn and MH1-MH4) homologous to Mal63p, a transcriptional activator for the genes involved in maltose utilization in Saccharomyces cerevisiae. The His478 to Leu substitution lay within the MH3 domain, corresponding to the negative regulatory domain of Mal63p which relieves the inhibitory effect on the activation function in response to maltose.
Abstract: A simple and rapid method for the preparation of a cell-free extract with the CCAAT-binding activity was established with Aspergillus nidulans as a model fungus. Proteins were extracted with 6 M guanidine hydrochloride directly from mycelia and renatured by dialysis. This method was found applicable to other filamentous fungi such as Aspergillus oryzae and Trichoderma viride.
Abstract: Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-beta-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species.
Abstract: The Taka-amylase A gene (taaG2) of Aspergillus oryzae is inducibly expressed in A. nidulans upon exposure to inducing carbon sources, such as starch and maltose. In order to identify nuclear factor(s) possibly involved in the induction of the taaG2 gene, gel mobility shift assays and DNase I footprinting analyses were carried out, and revealed a novel nuclear factor in A. nidulans extracts, which specifically bound to two sites in the taaG2 promoter region, -204 to -189 and -182 to -168, which share the common sequence GGAAATT. The nuclear factor was detected in nuclei from both induced and uninduced mycelia. Mutational analysis within and around the binding sequences demonstrated that only the upstream binding sequence, designated SRE (starch responsive element), was required for the inducible expression of the taaG2 gene, and thus we designated the nuclear factor SREB (SRE binding factor). The downstream binding site contained an inverted SRE (ISRE) and played no role in the induction of taaG2 expression. SREB was shown by gel retardation assays to have higher affinity for SRE than for ISRE.
Abstract: Regulatory CCAAT boxes are found frequently in eukaryotic promoter regions. They are bound by different CCAAT-binding factors. Until now, a single CCAAT-binding complex has been reported in fungi. It is also found in higher eukaryotes and is highly conserved among eukaryotic organisms. This multimeric protein complex is designated HAP, AnCF, CBF, or NF-Y. The complex consists of at least three subunits. In fungi, only the HAP complex of Saccharomyces cerevisiae had been known for a long time. The recent cloning of genes encoding the components of the corresponding complex (AnCF/PENR1) of Aspergillus nidulans and characterization of CCAAT-regulated genes in A. nidulans, as well as other filamentous fungi, led to a deeper insight into the role of this transcription complex, in particular in aerobically growing fungi. An overview of the function of HAP-like complexes in gene regulation in filamentous fungi is presented. Some of the genes that have been found to be regulated by HAP-like complexes encode enzymes of biotechnological interest, like taka-amylase, xylanases, cellobiohydrolase, and penicillin biosynthesis enzymes. The importance of HAP-like complexes in controlling the expression of biotechnologically important genes is discussed.
Abstract: Regulation of the Chaetomium gracile xylanase A gene (cgxA) was investigated using Aspergillus nidulans as an intermediate host. Deletion of a 185 bp DNA fragment from its promoter region led to higher levels of the cgxA gene expression, indicating that the 185 bp DNA fragment contains an element involved in repression of the gene. A nuclear extract was assayed for proteins which bind to the 185 bp DNA fragment. A protein designated AnRP bound sequence specifically to the DNA fragment. The minimum sequence required for AnRP binding, 5'TTGACAAAT-3', was determined by means of gel mobility shift assays with various double-stranded oligonucleotides. Furthermore, this sequence repressed the expression of the cgxA gene when inserted at the 5' end of the cgxA gene on pXAH, which was deleted for the repressive element from the promoter region.
Abstract: Endo-beta-1,4-glucanase A (EG A) of Aspergillus nidulans was purified to homogeneity, and its genomic gene (eglA) was cloned based on partial amino acid sequences of the purified enzyme and sequenced. The eglA gene comprised 1228 bp with four putative introns and encoded a polypeptide of 326 amino acids bearing high homology to the family A cellulases. The eglA promoter activity in A. nidulans was examined using the A. oryzae Taka-amylase A gene as a reporter. Expression of the reporter gene was induced by carboxymethylcellulose and cellobiose, and repressed by glucose, galactose, mannose, xylose, sorbitol, glycerol and succinate. Lactose neither induced nor repressed the expression.
Abstract: The Aspergillus nidulans hapC gene was expressed as a fusion protein with MalE or glutathione-S-transferase (GST) in Escherichia coli, and used for the purification of HapC and the preparation of anti-HapC antiserum. The CCAAT-binding factor AnCP/AnCF contains a component with an approximate molecular mass of 32 kDa that cross-reacts with the antibody. The MalE-HapC fusion protein was able to replace authentic HapC in AnCP when incubated under appropriate conditions. Furthermore, reconstitution experiments with recombinant HapC, yHAP2 and yHAP5 polypeptides showed that all three polypeptides were required for the assembly of a complex capable of binding to CCAAT-containing taaG2 promoter DNA. The relationship between AnCP/AnCF and the Saccharomyces cerevisiae HAP complex is discussed.
Abstract: A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.
Abstract: Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A. nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells.
Abstract: We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis.
Abstract: The N-terminal part of the CreA protein encompassing two zinc fingers was expressed in Escherichia coli as a fusion protein with the maltose binding protein (MalE) of E. coli. Our results show that CreA binds to the promoter of the Taa-G2 gene encoding Taka-amylase A of Aspergillus oryzae. DNase I footprinting experiments showed that CreA bound to three sites with high affinity and to one site with low affinity within the first 401-bp region upstream of the transcription initiation site. All of the sites contained sequences related to the CreA consensus binding site (5'-SYGGRG-3'), and are suggested to participate in repression of the Taa-G2 gene in response to glucose.
Abstract: We efficiently produced a small peptide by the host-vector system using Bacillus brevis as a host. DNA encoding the physiologically functional casoxin D, composed of seven amino acids, was ligated in tandem. An expression-secretion vector containing DNA, which codes for a fusion protein of epidermal growth factor-casoxin D pentamer, was constructed. B. brevis transformed with this plasmid produced about 0.5 g/liter of the fusion protein in the culture supernatant. The fusion protein was purified with ammonium sulfate fractionation from the supernatant and digested with two kinds of proteinases. A peptide well separated by high pressure liquid chromatography was identified as biologically active casoxin D.
Abstract: With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.
Abstract: Polypeptides comprising 20 amino acid residues (Y2) were covalently bound to the carboxyl terminus of a truncated proOmpA (proOmpA-D72C) through N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (X). The length of the inverted linker domain was 2.8 nm. proOmpA-D72C-X-Y2 thus synthesized was subjected to in vitro translocation into everted membrane vesicles of Escherichia coli. The conjugated protein was translocation-competent in terms of both proteinase K resistance and signal peptide cleavage, when a proton motive force (delta microH+) was imposed. The translocation was ATP-dependent. The proteinase K-treatment resulted in the digestion of SecA, SecE, and SecY in the membrane, suggesting that the proteinase K resistance of the Y2 domain was not due to its interaction with these Sec proteins in the secretory machinery. In the absence of delta microH+, the translocation ceased at the linker domain. Upon the imposition of delta microH+, the linker-Y2 domain underwent translocation, which did not require ATP hydrolysis as in the case of the translocation of the latter portion of usual secretory proteins. The translocation was prevented by anti-Y2 IgG even when delta microH+ was imposed. Another conjugated protein, which possesses a polypeptide comprising 61 amino acid residues after the linker (proOmpA-D72C-X-Lpp'), was synthesized. This compound was also translocated into everted membrane vesicles with cleavage of the signal peptide. These results suggest that substances to be translocated through the secretory machinery need not necessarily be solely held together by polypeptide bonds.
Abstract: The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.
Abstract: Expression of the ompF gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the ompF promoter. We previously demonstrated that this cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix. To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the ompF promoter were examined after they had been connected to the lacZ gene. The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the ompF promoter. It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the ompF expression. A local structural alteration in the ompF promoter was observed in some of the base-substitution mutants. Based on the results, the structure and function of the upstream sequence of the ompF promoter are discussed in relation to activation of the ompF promoter by the OmpR protein.
Abstract: The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.
Abstract: The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompC and ompF genes that specify the major outer membrane proteins OmpC and OmpF, respectively. We purified the OmpR protein not only from wild-type cells but also from two ompR mutants (ompR2 and ompR3) exhibiting quite different phenotypes as to osmoregulation of the ompC and ompF genes. The OmpR2 protein has an amino acid conversion in the C-terminal portion of the OmpR polypeptide, whereas the OmpR3 protein has one in the N-terminal portion. Comparative studies on these purified OmpR proteins were carried out in terms of their interaction with the ompC and ompF promoters. The nucleotide sequences involved in OmpR-binding were determined in individual promoter regions by deoxyribonuclease I footprinting. The OmpR3 protein as well as the wild-type OmpR protein appeared to bind, to similar extents, to both the ompC and ompF promoters. In contrast, the OmpR2 protein bound preferentially to the ompF promoter and failed to protect the ompC promoter against DNAse I digestion. These results support the view that the C-terminal portion of the OmpR protein is responsible for the binding of the OmpR protein to the ompC and ompF promoter DNAs. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.
Abstract: It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.
Abstract: OmpR protein is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. OmpR protein is considered to have a two-domain structure. In the present study we isolated a C-terminal fragment of the OmpR molecule, which bound to a specific promoter sequence of the ompF gene. We conclude that the C-terminal portion of the OmpR protein contains a DNA-binding site.
