Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. Conclusions Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.University of Murcia and University of Seville was supported by projects: BIO2015-63949-R, BIO2014-54411-C2-1-REuropa MINECO/FEDER RTI2018-094393-B-C21Fundación Séneca (Grant no. 19236/PI/14

Structure and non-essential function of glycerol kinase in <i>Plasmodium</i> <i>falciparum</i> blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito

Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans

Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus nidulans conidia and rapidly degraded upon induction of conidial germination. Furthermore, trehalose is accumulated in response to a heat shock or to an oxidative shock. The authors have characterized the A. nidulans tpsA gene encoding trehalose-6-phosphate synthase, which catalyses the first step in trehalose biosynthesis. Expression of tpsA in a Saccharomyces cerevisiae tps1 mutant revealed that the tpsA gene product is a functional equivalent of the yeast Tps1 trehalose-6-phosphate synthase. The A. nidulans tpsA-null mutant does not produce trehalose during conidiation or in response to various stress conditions. While germlings of the tpsA mutant show an increased sensitivity to moderate stress conditions (growth at 45 °C or in the presence of 2 mM H2O2), they display a response to severe stress (60 min at 50 °C or in the presence of 100 mM H2O2) similar to that of wild-type germlings. Furthermore, conidia of the tpsA mutant show a rapid loss of viability upon storage. These results are consistent with a role of trehalose in the acquisition of stress tolerance. Inactivation of the tpsA gene also results in increased steady-state levels of sugar phosphates but does not prevent growth on rapidly metabolizable carbon sources (glucose, fructose) as seen in Saccharomyces cerevisiae. This suggests that trehalose 6-phosphate is a physiological inhibitor of hexokinase but that this control is not essential for proper glycolytic flux in A. nidulans. Interestingly, tpsA transcription is not induced in response to heat shock or during conidiation, indicating that trehalose accumulation is probably due to a post-translational activation process of the trehalose 6-phosphate synthase

Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

10.1371/journal.pone.0056037PLoS ONE82

LC-MS proteomics analysis of the iInsulin/IGF-1-deficient Caenorhabditis elegans daf-2(e1370) mutant reveals extensive restructuring of intermediary metabolism

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC-MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid beta-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity

The gene YALI0E20207g from Yarrowia lipolytica encodes an N-acetylglucosamine kinase implicated in the regulated expression of the genes from the N-acetylglucosamine assimilatory pathway

This is an open access article distributed under the terms of the Creative Commons Attribution License.The non-conventional yeast Yarrowia lipolyticapossesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases fromdifferent organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion.We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway - identified by a BLAST search - was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1 glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose.This work was supported by grant BFU2010-19628-C02-02 from the Spanish Ministry for Science and Innovation (MICINN, now MINECO).Peer Reviewe

Cosmid Cloning of Five \u3cem\u3eZymomonas trp\u3c/em\u3e Genes by Complementation of \u3cem\u3eEscherichia coli\u3c/em\u3e and \u3cem\u3ePseudomonas putida trp\u3c/em\u3e Mutants

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes