Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants

<p>Abstract</p> <p>Background</p> <p>Most bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by <it>ptsG, mgsA </it>mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.</p> <p>Results</p> <p>In order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h^-1using wild type <it>Escherichia coli</it>. The result indicates that the transcript levels of global regulators such as <it>crp, cra, mlc </it>and <it>rpoS </it>decreased, while those of <it>fadR, iclR, soxR/S </it>increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO₂evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the ¹³C-flux analysis. In order to make clear the catabolite regulation, the effect of <it>crp </it>gene knockout (Δ<it>crp</it>) and crp enhancement (<it>crp⁺</it>) as well as <it>mlc, mgsA, pgi </it>and <it>ptsG </it>gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h^-1and by some batch cultures. In the case of Δ<it>crp </it>(and also Δ<it>mlc</it>) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of <it>crp⁺</it>mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of <it>ptsG </it>and <it>ptsH </it>by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by <it>mgsA, pgi</it>, and <it>ptsG </it>mutants due to increase in <it>crp </it>as well as <it>cyaA</it>, while glucose consumption rate became lower.</p> <p>Conclusions</p> <p>The transcriptional catabolite regulation mechanism was made clear for the wild type <it>E. coli</it>, and its <it>crp, mlc, ptsG, pgi, and mgsA </it>gene knockout mutants. The results indicate that catabolite repression can be relaxed and <it>crp </it>as well as <it>cyaA </it>can be increased by <it>crp⁺, mgsA, pgi</it>, and <it>ptsG </it>mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of <it>ptsG </it>in all the mutants considered.</p

Upregulation of Aquaporin-3 Is Involved in Keratinocyte Proliferation and Epidermal Hyperplasia

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed in keratinocytes of the epidermis. We previously showed that AQP3-mediated transport of water and glycerol is involved in keratinocyte migration and proliferation, respectively. However, the involvement of AQP3 in epidermal hyperplasia in skin diseases, such as atopic dermatitis (AD), is unknown. In this study, we found significantly increased AQP3 transcript and protein expression in the epidermis of human AD lesions. The upregulation of AQP3 expression in human keratinocytes by transfection with human AQP3 DNA plasmid was associated with increased cellular glycerol and ATP, as well as increased cell proliferation. Among several cytokines and chemokines produced in the skin, CCL17, which is highly expressed in AD, was found to be a strong inducer of AQP3 expression and enhanced keratinocyte proliferation. In mouse AD models, AQP3 was strongly overexpressed in the epidermis in wild-type mice. Epidermal hyperplasia was reduced in AQP3-deficient mice, with a decreased number of proliferating keratinocytes. These results suggest the involvement of AQP3 in epidermal hyperplasia by a mechanism involving upregulated AQP3 expression and consequent enhancement of keratinocyte proliferation

Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks

Update on the Keio collection of Escherichia coli single-gene deletion mutants

The Keio collection (Baba et al, 2006) has been established as a set of single‐gene deletion mutants of Escherichia coli K‐12. These mutants have a precisely designed deletion from the second codon from the seventh to the last codon of each predicted ORF. Further information is available at http://sal.cs.purdue.edu:8097/GB7/index.jsp or http://ecoli.naist.jp/. The distribution is now being handled by the National Institute of Genetics of Japan (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp). To date more than 4 million samples have been distributed worldwide. As we described earlier (Baba et al, 2006), gene amplification during construction is likely to have led to a small number of mutants with genetic duplications

Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>In <it>Escherichia coli</it>, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of <it>E. coli </it>with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.</p> <p>Results</p> <p>We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three <it>cis</it>-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.</p> <p>Conclusions</p> <p>This comprehensive screen for <it>E. coli </it>sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the <it>E. coli </it>genome. We constructed the <it>Escherichia coli </it>Small RNA Browser (ECSBrowser; <url>http://rna.iab.keio.ac.jp/</url>), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study.</p

Reduction of Skin pH during Treatment for Palmoplantar Hyperhidrosis: A Conjecture on the Role of pH-Regulated Water Channel, i.e. Aquaporin

Primary palmoplantar hyperhidrosis (PPH) is a disorder that involves excessive sweating on the palms and soles. Although the pathophysiology of PPH remains unknown, some treatments, including topical aluminum chloride (AC) and tap water iontophoresis (TWI), are effective at suppressing the perspiration. Herein, we report the kinetics of the skin pH of two cases of PPH treated with AC and TWI. We found that the skin pH decreased in accordance with the reduction in sweating. This finding indicates that the reduction in sweating may be attributed to the reduction of skin pH in AC and TWI. Whether or not the pH-regulated function of aquaporin can explain this finding remains unknown

Normal Immunostaining Pattern for Aquaporin-5 in the Lesions of Palmoplantar Hyperhidrosis

Aquaporin-5 (AQP-5) is a water-transporting protein expressed in mammal sweat glands. It has been reported that the expression of AQP-5 is involved in sweating of mice, rats, and horses. However, the physiological function of human AQP-5 is still uncertain. In this report, we examined the expression pattern of AQP-5 in the skin lesions of palmoplantar hyperhidrosis in patients with Nagashima-type palmoplantar hyperkeratosis (PPK). We found that there was no significant difference in AQP-5 expression in the palmoplantar skin of healthy subjects and patients with palmoplantar hyperhidrosis. Our findings suggest that a mechanism other than AQP-5 may be involved in the pathogenesis of hyperhidrosis in PPK