Dataset Information

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

ABSTRACT: Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

SUBMITTER: Torres JC

PROVIDER: S-EPMC1218843 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.

Dataset Information

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets