Computer‐aided rational design of the phosphotransferase system for enhanced glucose uptake inEscherichia coli

ABSTRACT Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyperproducing mutant, were examined during various stages of growth. Glucose uptake in C. beijerinckii BA101 was repressed 20% by 2-deoxyglucose and 25% by mannose, while glucose uptake in C. beijerinckii8052 was repressed 52 and 28% by these sugars, respectively. We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts ofC. beijerinckii BA101 by glucose phosphorylation by PEP. The PTS activity associated with C. beijerinckii BA101 was 50% of that observed for C. beijerinckii 8052.C. beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol. Glucose phosphorylation by cell extracts derived from both C. beijerinckii BA101 and 8052 was also dependent on the presence of ATP, a finding consistent with the presence of glucokinase activity in C. beijerinckii extracts. ATP-dependent glucose phosphorylation was predominant during the solventogenic stage, when PEP-dependent glucose phosphorylation was dramatically repressed. A nearly twofold-greater ATP-dependent phosphorylation rate was observed for solventogenic stage C. beijerinckii BA101 than for solventogenic stage C. beijerinckii 8052. These results suggest that C. beijerinckii BA101 is defective in PTS activity and that C. beijerinckii BA101 compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage.

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Proteomic and Transcriptomic Elucidation of the MutantRalstonia eutrophaG+1 with Regard to Glucose Utilization

Applied and Environmental Microbiology ◽

10.1128/aem.02015-10 ◽

2011 ◽

Vol 77 (6) ◽

pp. 2058-2070 ◽

Cited By ~ 32

Author(s):

Matthias Raberg ◽

Katja Peplinski ◽

Silvia Heiss ◽

Armin Ehrenreich ◽

Birgit Voigt ◽

...

Keyword(s):

Glucose Uptake ◽

Ralstonia Eutropha ◽

Stationary Growth Phase ◽

Insertion Mutation ◽

Secondary Effects ◽

Phosphotransferase System ◽

Stationary Growth ◽

Increase Glucose Uptake ◽

Mutant Cells ◽

Intracellular Glucose

ABSTRACTBy taking advantage of the available genome sequence ofRalstonia eutrophaH16, glucose uptake in the UV-generated glucose-utilizing mutantR. eutrophaG+1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usuallyN-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encodingnagoperon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation innagE(membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutantR. eutrophaG+1 ΔnagFECto utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-β-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.

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Optimal Sensor Design Using Patient-Specific Images

Journal of Medical Devices ◽

10.1115/1.3443743 ◽

2010 ◽

Vol 4 (2) ◽

Author(s):

Sukhi Basati ◽

Timothy J. Harris ◽

Andreas A. Linninger

Keyword(s):

Computer Aided Design ◽

Rational Design ◽

Treatment Options ◽

Patient Specific ◽

Data Sets ◽

Disease States ◽

Magnetic Resonance Data ◽

Computer Aided ◽

Aided Design ◽

Pathological Disease

In diseases such as hydrocephalus, the cerebral ventricles enlarge. The treatment options for these patients are presently based on pressure, which has limited capabilities. We present the design of a volume sensor as an alternative monitoring option. Through the use of computer aided design and simulation, we optimized a sensor in silico with fewer resources. Specifically, we designed a sensor for animal experimentation with a scalable procedure for human sensors. In this paper, we present a rational design approach for a sensor that integrates advances in medical imaging. Magnetic resonance data sets of both normal and diseased subjects were used as a virtual laboratory. Finite element simulations were performed under pathological disease states of the brain as a contribution toward an accelerated device design. An optimized sensor was then fabricated for these subjects based on the outcome of the simulations. In this paper, we explain how a computer aided subject-specific design was used to help fabricate and test our sensor.

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Phosphoenolpyruvate-Dependent Glucose Transport in Oral Streptococci

Journal of Dental Research ◽

10.1177/00220345730520060801 ◽

1973 ◽

Vol 52 (6) ◽

pp. 1209-1215 ◽

Cited By ~ 37

Author(s):

Charles F. Schachtele ◽

John A. Mayo

Keyword(s):

Glucose Uptake ◽

Streptococcus Mutans ◽

Glucose Transport ◽

Enzyme System ◽

Cell Suspensions ◽

Oral Streptococci ◽

Phosphotransferase System ◽

Resting Cell

Streptococcus mutans, S sanguis, and S salivarius use a phosphoenolpyruvate (PEP)-dependent phosphotransferase system that results in phosphorylation of glucose at carbon 6. This enzyme system is not sensitive to fluoride. Glucose uptake into resting cell suspensions is sensitive to fluoride because of inhibition of intracellular PEP production. The glucose phosphotransferase system is constitutive in oral streptococci.

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