Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures

Background: It is important to understand the cellular responses emanating from environmental perturbations to redesign the networks for practical applications. In particular, the carbon (C) metabolism, nitrogen (N) assimilation, and energy generation are by far important, where those are interconnected and integrated to maintain cellular integrity. In our previous study, we investigated the effect of C/N ratio on the metabolic regulation of gdhA, glnL, glt B,D mutants as well as wild type Escherichia coli (Kumar and Shimizu, MCF, 1-17, 9:8,2010), where it was shown that the transcript levels of cyoA and cydB which encode the terminal oxidases, fnr and fur which encode global regulators were significantly up-regulated under N-limited condition as compared to C-limited condition. In the present study, therefore, the effects of such single-gene knockout on the metabolic regulation were investigated to clarify the roles of those genes in the aerobic continuous culture at the dilution rate of 0.2 h(-1). Results: The specific glucose consumption rates and the specific CO2 production rates of cyoA, cydB, fnr, and fur mutants were all increased as compared to the wild type under both C-limited and N-limited conditions. The former phenomenon was consistent with the up-regulations of the transcript levels of ptsG and ptsH, which are consistent with down-regulations of crp and mlc genes. Moreover, the increase in the specific glucose consumption rate was also caused by up-regulations of the transcript levels of pfkA, pykF and possibly zwf, where those are consistent with the down regulations of cra, crp and mlc genes. Moreover, the transcript levels of rpoN together with glnK, glnB, glnE were up-regulated, and thus the transcript levels of glnA, L, G, and gltB,D as well as nac were up-regulated, while gdhA was down-regulated. This implies the interconnection between cAMP-Crp and PII-Ntr systems. Moreover, cyoA, cydB, fnr and fur gene deletions up-regulated the transcript levels of respiration (nuoA, ndh, cyoA, cydB, and atpA) and the oxidative stress related genes such as soxR, S and sodA, where this was further enhanced under N-limitation. In the cases of cyoA and cydB mutants, arcA, fnr, fur, cydB (for cyoA mutant), and cyoA (for cydB mutant) genes were up-regulated, which may be due to incomplete oxidation of quinol. It was also shown that fur gene transcript level was up-regulated in accordance with the activation of respiratory chain genes. It was shown that the deletion of fur gene activated the enterobactin pathway. Conclusion: The present result demonstrated how the fermentation characteristics could be explained by the transcript levels of metabolic pathway genes as well as global regulators in relation to the knockout of such single genes as cyoA, cydB, fnr, and fur, and clarified the complex gene network regulation in relation to glycolysis, TCA cycle, respiration, and N-regulated pathways. The present result is quite important in understanding the metabolic regulation for metabolic engineering. Moreover, the present result may be useful in improving the specific glucose consumption rate and activation of the TCA cycle by modulating the respiratory chain genes and the related global regulators. The result obtained under N-limited condition may be useful for the heterologous protein production under N-limitation

Metabolic regulation of Escherichia coli and its phoB and phoR genes knockout mutants under phosphate and nitrogen limitations as well as at acidic condition

<p>Abstract</p> <p>Background</p> <p>The phosphorus compounds serve as major building blocks of many biomolecules, and have important roles in signal transduction. The phosphate is involved in many biochemical reactions by the transfer of phosphoryl groups. All living cells sophisticatedly regulate the phosphate uptake, and survive even under phosphate-limiting condition, and thus phosphate metabolism is closely related to the diverse metabolism including energy and central carbon metabolism. In particular, phosphorylation may play important roles in the metabolic regulation at acidic condition and nitrogen limiting condition, which typically appears at the late growth phase in the batch culture. Moreover, phosphate starvation is a relatively inexpensive means of gene induction in practice, and the <it>phoA </it>promoter has been used for overexpression of heterologous genes. A better understanding of phosphate regulation would allow for optimization of such processes.</p> <p>Results</p> <p>The effect of phosphate (P) concentration on the metabolism in <it>Escherichia coli </it>was investigated in terms of fermentation characteristics and gene transcript levels for the aerobic continuous culture at the dilution rate of 0.2 h^-1. The result indicates that the specific glucose consumption rate and the specific acetate production rate significantly increased, while the cell concentration decreased at low P concentration (10% of the M9 medium). The increase in the specific glucose uptake rate may be due to ATP demand caused by limited ATP production under P-limitation. The lower cell concentration was also caused by less ATP production. The less ATP production by H⁺-ATPase may have caused less cytochrome reaction affecting in quinone pool, and caused up-regulation of ArcA/B, which repressed TCA cycle genes and caused more acetate production. In the case of <it>phoB </it>mutant (and also <it>phoR </it>mutant), the fermentation characteristics were less affected by P-limitation as compared to the wild type where the PhoB regulated genes were down-regulated, while <it>phoR </it>and <it>phoU </it>changed little. The <it>phoR </it>gene knockout caused <it>phoB </it>gene to be down-regulated as well as PhoB regulated genes, while <it>phoU </it>and <it>phoM </it>changed little. The effect of pH together with lower P concentration on the metabolic regulation was also investigated. In accordance with up-regulation of <it>arcA </it>gene expression, the expressions of the TCA cycle genes such as <it>sdhC </it>and <it>mdh </it>were down-regulated at acidic condition. The gene expression of <it>rpoS </it>was up-regulated, and the expression of <it>gadA </it>was up-regulated at pH 6.0. In accordance with this, PhoB regulated genes were up-regulated in the wild type under P-rich and P-limited conditions at pH 6.0 as compared to those at pH 7.0. Moreover, the effect of nitrogen limitation on the metabolic regulation was investigated, where the result indicates that <it>phoB </it>gene was up-regulated, and PhoB regulated genes were also up-regulated under N-limitation, as well as nitrogen-regulated genes.</p> <p>Conclusion</p> <p>The present result shows the complicated nature of the metabolic regulation for the fermentation characteristics upon phosphate limitation, acidic condition, and nitrogen limitation based on the transcript levels of selected genes. The result implies that the regulations under phosphate limitation, acidic condition, and nitrogen limitation, which occur typically at the late growth phase of the batch culture, are interconnected through RpoS and RpoD together with Pho genes.</p

Effect of cra Gene Mutation on the Metabolism of Escherichia Coli for a Mixture of Multiple Carbon Sources

The major player for catabolite repression is the phosphotransferase systems (PTSs) and cAMP-Crp. Moreover, Cra controls the carbon flow in the metabolic network. In the present research, the effect of modulating cra gene (Δcra) on the consumption of multiple carbon sources such as glucose and fructose (as well as xylose) was investigated under both aerobic and anaerobic conditions. It was shown that glucose and fructose could be co-metabolized with fructose consumed faster than glucose in cra mutant under both aerobic and anaerobic conditions. It was also implied that cra mutant consumed higher amount of total carbon sources, which contributed to the highest metabolite production as compared to the wild type strain. Thus, cra mutant can be a good candidate for the efficient utilization of multiple carbon sources such as glucose and fructose, where xylose consumption was repressed by catabolite repression. The overall regulation mechanisms were clarified based on fermentation data and gene transcript analysis

Effect of temperature up-shift on fermentation and metabolic characteristics in view of gene expressions in Escherichia coli

Abstract Background Escherichia coli induces heat shock genes to the temperature up-shift, and changes the metabolism by complicated mechanism. The heat shock response is of practical importance for the variety of applications such as temperature-induced heterologous protein production, simultaneous saccharification and fermentation (SSF) etc. However, the effect of heat shock on the metabolic regulation is not well investigated. It is strongly desired to understand the metabolic changes and its mechanism upon heat shock in practice for the efficient metabolite production by temperature up-shift. In the present research, therefore, we investigated the effect of temperature up-shift from 37°C to 42°C on the metabolism in view of gene expressions. Results The results of aerobic batch and continuous cultivations of E. coli BW25113 indicate that more acetate was accumulated with lower biomass yield and less glucose consumption rate at 42°C as compared to the case at 37°C. The down- regulation of the glucose uptake rate corresponds to the down-regulation of ptsG gene expression caused by the up-regulation of mlc gene expression. In accordance with up-regulation of arcA, which may be caused by the lower oxygen solubility at 42°C, the expressions of the TCA cycle-related genes and the respiratory chain gene cyoA were down-regulated. The decreased activity of TCA cycle caused more acetate formation at higher temperature, which is not preferred in heterologous protein production etc. This can be overcome by the arcA gene knockout to some extent. The time courses of gene expressions revealed that the heat shock genes such as groEL, dnaK, htpG and ibpB as well as mlc were expressed in much the same way as that of rpoH during the first 10–20 minutes after temperature up-shift. Under microaerobic condition, the fermentation changed in such a way that formate and lactate were more produced due to up-regulation of pflA and ldhA genes while ethanol was less produced due to down-regulation of adhE gene at higher temperature as compared to the case at 37°C. Conclusion The present result clarified the mechanism of metabolic changes upon heat shock from 37°C to 42°C based on gene expressions of heat shock genes, global regulators, and the metabolic pathway genes. It is recommended to use arcA gene knockout mutant to prevent higher acetate production upon heat shock, where it must be noted that the cell yield may be decreased due to TCA cycle activation by arcA gene knockout.</p

Determination of the structure of 31^{31}Ne by full-microscopic framework

We perform the first quantitative analysis of the reaction cross sections of $^{28-32}$Ne by $^{12}$C at 240 MeV/nucleon, using the double-folding model (DFM) with the Melbourne $g$-matrix and the deformed projectile density calculated by the antisymmetrized molecular dynamics (AMD). To describe the tail of the last neutron of $^{31}$Ne, we adopt the resonating group method (RGM) combined with AMD. The theoretical prediction excellently reproduce the measured cross sections of $^{28-32}$Ne with no adjustable parameters. The ground state properties of $^{31}$Ne, i.e., strong deformation and a halo structure with spin-parity $3/2_{}^-$, are clarified.Comment: 4 pages, 4 figures, 2 table

Integration of enzyme activities into metabolic flux distributions by elementary mode analysis

<p>Abstract</p> <p>Background</p> <p>In systems biology, network-based pathway analysis facilitates understanding or designing metabolic systems and enables prediction of metabolic flux distributions. Network-based flux analysis requires considering not only pathway architectures but also the proteome or transcriptome to predict flux distributions, because recombinant microbes significantly change the distribution of gene expressions. The current problem is how to integrate such heterogeneous data to build a network-based model.</p> <p>Results</p> <p>To link enzyme activity data to flux distributions of metabolic networks, we have proposed Enzyme Control Flux (ECF), a novel model that integrates enzyme activity into elementary mode analysis (EMA). ECF presents the power-law formula describing how changes in enzyme activities between wild-type and a mutant are related to changes in the elementary mode coefficients (EMCs). To validate the feasibility of ECF, we integrated enzyme activity data into the EMCs of <it>Escherichia coli </it>and <it>Bacillus subtilis </it>wild-type. The ECF model effectively uses an enzyme activity profile to estimate the flux distribution of the mutants and the increase in the number of incorporated enzyme activities decreases the model error of ECF.</p> <p>Conclusion</p> <p>The ECF model is a non-mechanistic and static model to link an enzyme activity profile to a metabolic flux distribution by introducing the power-law formula into EMA, suggesting that the change in an enzyme profile rather reflects the change in the flux distribution. The ECF model is highly applicable to the central metabolism in knockout mutants of <it>E. coli </it>and <it>B. subtilis</it>.</p

Time-Series Analysis of Video Comments on Social Media

In this study, we propose a method to detect unfair rating cheat caused by multiple comment postings focusing on time-series analysis of the number of comments. We defined the videos that obtained a lot of comments by unfair cheat as ‘unfair video’ and defined the videos which obtained without unfair cheat as ‘popular video’. Specifically, our proposed method focused on the difference of chronological distributions of the comments between the popular videos and the unfair videos. As the evaluation result, our proposed method could obtain higher accuracy than that of the baseline method