Energy coupling to the transport of inorganic phosphate in Escherichia coli K12.
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ABSTRACT:
SUBMITTER: Rosenberg H
PROVIDER: S-EPMC1186489 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:1. Anaerobic uptake of proline requires either the presence of a coupled Mg2+-stimulated adenosine triphosphatase or anaerobic electron transport. 2. Anaerobic uptake of glutamine does not require anaerobic electron transport even in the absence of a coupled Mg+2-stimulated adenosine triphosphatase. 3. These results support previous suggestions [Berger (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514--1518; Berger & Heppel (1974) J. Biol. Chem. 249, 7747-7755; Kobayashi, Kin & Anraku (1974) J. Biochem. (Tokyo) 76, 251-261] that two distinct mechanisms of energy coupling to active transport exist in Escherichia coli in that energization of anaerobic proline uptake requires the 'high-energy membrane state', whereas the energization of anaerobic glutamine uptake does not.
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Project description:In E. coli the phosphate homeostasis is regulated by the Pst system and the two-component system PhoB/R. Pathogens like E. coli O157:H7 are responsible for many outbreaks and can be found and survive in poor inorganic phosphate (Pi) environments. To understand global EHEC O157:H7 EDL933 strain responses to Pi-starvation, we compared the transcriptomes of EDL933 the WT strain grown in MOPS Pi rich medium and that grown in MOPS Pi poor medium, using the Affymetrix GeneChip® E. coli Genome 2.0 Array. Also we investigated the EDL933 global response to the absence of PhoB by comparing the transcriptomes of the WT strain the ΔphoB mutant both grown in Low Pi
2014-03-15 | GSE50529 | GEO
Project description:BackgroundIn the L-histidine (His) biosynthetic pathway of Escherichia coli, the first key enzyme, ATP-phosphoribosyltransferase (ATP-PRT, HisG), is subject to different types of inhibition. Eliminating the feedback inhibition of HisG by the His end product is an important step that enables the oversynthesis of His in breeding strains. However, the previously reported feedback inhibition-resistant mutant enzyme from E. coli, HisGE271K, is inhibited by purine nucleotides, particularly ADP and AMP, via competitive inhibition with its ATP substrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is formed not only during His biosynthesis but also during de novo purine biosynthesis, acts as a natural analog of AMP and substitutes for it in some enzymatic reactions. We hypothesized that AICAR could control its own formation, particularly through the His biosynthetic pathway, by negatively influencing HisG enzymatic activity, which would make preventing ATP-PRT transferase inhibition by AICAR crucial for His overproduction.ResultsFor the first time, both the native E. coli HisG and the previously described feedback-resistant mutant HisGE271K enzymes were shown to be sensitive to inhibition by AICAR, a structural analog of AMP. To circumvent the negative effect that AICAR has on His synthesis, we constructed the new His-producing strain EA83 and demonstrated its improved histidine production. This increased production was particularly associated with the improved conversion of AICAR to ATP due to purH and purA gene overexpression; additionally, the PitA-dependent phosphate/metal (Me2+-Pi) transport system was modified by a pitA gene deletion. This His-producing strain unexpectedly exhibited decreased alkaline phosphatase activity at low Pi concentrations. AICAR was consequently hypothesized inhibit the two-component PhoBR system, which controls Pho regulon gene expression.ConclusionsInhibition of a key enzyme in the His biosynthetic pathway, HisG, by AICAR, which is formed in this pathway, generates a serious bottleneck during His production. The constructed His-producing strain demonstrated the enhanced expression of genes that encode enzymes involved in the metabolism of AICAR to ATP, which is a substrate of HisG, and thus led to improved His accumulation.
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Project description:Enterohemorrhagic Escherichia coli (EHEC), an emerging food- and water-borne hazard, is highly pathogenic to humans. In the environment, EHEC must survive phosphate (Pi) limitation. The response to such Pi starvation is an induction of the Pho regulon including the Pst system that senses Pi variation. The interplay between the virulence of EHEC, Pho-Pst system and environmental Pi remains unknown. To understand the effects of Pi deprivation on the molecular mechanisms involved in EHEC survival and virulence under Pho regulon control, we undertook transcriptome profiling of the EDL933 wild-type strain grown under high Pi and low Pi conditions and its isogenic ΔphoB mutant grown in low Pi conditions. The differentially expressed genes included 1067 Pi-dependent genes and 603 PhoB-dependent genes. Of these 131 genes were both Pi and PhoB-dependent. Differentially expressed genes that were selected included those involved in Pi homeostasis, cellular metabolism, acid stress, oxidative stress and RpoS-dependent stress responses. Differentially expressed virulence systems included the locus of enterocyte effacement (LEE) encoding the type-3 secretion system (T3SS) and its effectors, as well as BP-933W prophage encoded Shiga toxin 2 genes. Moreover, PhoB directly regulated LEE and stx2 gene expression through binding to specific Pho boxes. However, in Pi-rich medium, constitutive activation of the Pho regulon decreased LEE gene expression and reduced adherence to HeLa cells. Together, these findings reveal that EHEC has evolved a sophisticated response to Pi limitation involving multiple biochemical strategies that contribute to its ability to respond to variations in environmental Pi and to coordinating the virulence response.
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Project description:BACKGROUND:Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into gamma-aminobutyrate. But genes coding for these enzymes have not been identified so far. RESULTS:The 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains sigma54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the sigma54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29). The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0. CONCLUSION:Expression of E. coli K12 ygjG coding region revealed sigma54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.
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Project description:DNA microarrays were conducted on E. coli K12 cells stressed with 10 μM in N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Overall, 260 genes varied in expression, 114 up-regulated and 146 down-regulated by Zn deprivation Keywords: TPEN stress
2008-12-31 | GSE11303 | GEO
Project description:1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg(2+)+Ca(2+)-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.
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Project description:Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence.
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Project description:Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.
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