scholarly journals Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment

2003 ◽  
Vol 69 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Maria-Manuel Sampaio ◽  
Helena Santos ◽  
Winfried Boos
Keyword(s):  
Escherichia Coli ◽  
Cold Water ◽  
Genetically Engineered ◽  
Isotopic Enrichment ◽  
Rhodothermus Marinus ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

ABSTRACT We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-α-d-mannosyl-d-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-α-d-mannosyl-d-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-α-d-mannosyl-d-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-α-d-mannosyl-d-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.

scholarly journals Escherichia coli Peptidase A, B, or N Can Process Translation Inhibitor Microcin C

2008 ◽  
Vol 190 (7) ◽  
pp. 2607-2610 ◽  
Author(s):  
Teymur Kazakov ◽  
Gaston H. Vondenhoff ◽  
Kirill A. Datsenko ◽  
Maria Novikova ◽  
Anastasia Metlitskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Trna Synthetase ◽  
Methionine Residue ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Translation Inhibitor ◽  
Rate Limiting ◽  
Broad Specificity

ABSTRACT The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.

scholarly journals Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose

1976 ◽  
Vol 156 (2) ◽  
pp. 477-480 ◽  
Author(s):  
D Herbert ◽  
H L Kornberg
Keyword(s):  
Escherichia Coli ◽  
Continuous Culture ◽  
Glucose Transport ◽  
Growth Rates ◽  
Glucose Limitation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Wide Range ◽  
Rate Limiting

Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.

scholarly journals The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors

1982 ◽  
Vol 203 (2) ◽  
pp. 505-510 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Maximum Velocity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Cytochrome C Reduction ◽  
Pattern Of Variation ◽  
Rate Limiting

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

scholarly journals Kaleidoscope

2014 ◽  
Vol 205 (4) ◽  
pp. 334-335
Author(s):  
Derek K. Tracy ◽  
Dan W. Joyce ◽  
Sukhwinder S. Shergill
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Genetic Variant ◽  
Age At Onset ◽  
Epidemiological Data ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
The Impact ◽  
Rate Limiting

It cannot have escaped our readers' notice that there has been a public increase in the awareness of the impact of dementia on people's lives: politicians have raised concerns about a dementia ‘time bomb’ as a greater number of people live to an older age; and even the 2013 G8 summit declared1 that there was a need for international initiatives to tackle this illness. The inevitable call for more research is underscored by the lack of any new licenced medications for Alzheimer's disease since 2002. There has been much interest in a putative role for statins – which inhibit the HMGCR enzyme, the rate-limiting step in cholesterol production – as retrospective epidemiological data have shown that they can reduce the risk of developing Alzheimer's disease by up to 70%; but, frustratingly, administration of these drugs to those with the illness appears to produce little benefit. Recent data have now shown that the gene encoding this HMGCR enzyme is a potent modifier for the age at onset and rate of conversion from mild cognitive impairment to Alzheimer's disease.2 Indeed, this work would indicate that its G-negative allele is second only to APOE2 as the most common and important protective genetic variant for spontaneous Alzheimer's disease.

scholarly journals Xanthomonas translucens commandeers the host rate-limiting step in ABA biosynthesis for disease susceptibility

2019 ◽  
Vol 116 (42) ◽  
pp. 20938-20946 ◽  
Author(s):  
Zhao Peng ◽  
Ying Hu ◽  
Junli Zhang ◽  
Jose C. Huguet-Tapia ◽  
Anna K. Block ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Disease Susceptibility ◽  
Susceptibility Gene ◽  
Abiotic Stress Response ◽  
Transcription Activator ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Xanthomonas Translucens ◽  
Rate Limiting

Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1. The results represent an appropriation of host physiology by a bacterial virulence effector.

scholarly journals Transport of d-Xylose in Lactobacillus pentosus, Lactobacillus casei, andLactobacillus plantarum: Evidence for a Mechanism of Facilitated Diffusion via the Phosphoenolpyruvate:Mannose Phosphotransferase System

1999 ◽  
Vol 181 (16) ◽  
pp. 4768-4773 ◽  
Author(s):  
Stéphane Chaillou ◽  
Peter H. Pouwels ◽  
Pieter W. Postma
Keyword(s):  
Dry Weight ◽  
Facilitated Diffusion ◽  
Xylose Metabolism ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Lactobacillus Pentosus ◽  
Xylose Transport ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Kinetics Of

ABSTRACT We have identified and characterized the d-xylose transport system of Lactobacillus pentosus. Uptake ofd-xylose was not driven by the proton motive force generated by malolactic fermentation and required d-xylose metabolism. The kinetics of d-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent Km of 8.5 mM and aV max of 23 nmol min−1 mg of dry weight−1. In two mutants of L. pentosusdefective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on d-xylose was absent due to the lack ofd-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIBMan component of the EIIMancomplex. The EIIMan complex is also involved ind-xylose transport in L. casei ATCC 393 andL. plantarum 80. These two species could transport and metabolize d-xylose after transformation with plasmids which expressed the d-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-d-glucose were defective in EIIMan activity and were unable to transportd-xylose when transformed with plasmids containing thexylAB genes. Finally, transport of d-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. caseiATCC 393 containing plasmids coding for thed-xylose-catabolic enzymes, since the doubling time of these bacteria on d-xylose was proportional to the level of EIIMan activity.

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