Pyridoxal and α-ketoglutarate independently improve function of Saccharomyces cerevisiae Thi5 in the metabolic network of Salmonella enterica

2021 ◽  
Author(s):  
Michael D. Paxhia ◽  
Diana M. Downs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Network ◽  
Legionella Pneumophila ◽  
Salmonella Enterica ◽  
Metabolic Flux ◽  
Genetic Suppressors ◽  
Metabolite Pool ◽  
Improved Function ◽  
Dependent Pathway

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica . Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report a strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica . Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica . IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contribute to their functional differences in a Salmonella enterica host.

scholarly journals The Response to 2-Aminoacrylate Differs in Escherichia coli and Salmonella enterica, despite Shared Metabolic Components

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Andrew J. Borchert ◽  
Diana M. Downs
Keyword(s):  
Escherichia Coli ◽  
Metabolic Network ◽  
Salmonella Enterica ◽  
Metabolic Networks ◽  
Metabolic Potential ◽  
Content Type ◽  
E Coli ◽  
Sense And Respond ◽  
External Stimuli

ABSTRACT The metabolic network of an organism includes the sum total of the biochemical reactions present. In microbes, this network has an impeccable ability to sense and respond to perturbations caused by internal or external stimuli. The metabolic potential (i.e., network structure) of an organism is often drawn from the genome sequence, based on the presence of enzymes deemed to indicate specific pathways. Escherichia coli and Salmonella enterica are members of the Enterobacteriaceae family of Gram-negative bacteria that share the majority of their metabolic components and regulatory machinery as the “core genome.” In S. enterica, the ability of the enamine intermediate 2-aminoacrylate (2AA) to inactivate a number of pyridoxal 5′-phosphate (PLP)-dependent enzymes has been established in vivo. In this study, 2AA metabolism and the consequences of its accumulation were investigated in E. coli. The data showed that despite the conservation of all relevant enzymes, S. enterica and E. coli differed in both the generation and detrimental consequences of 2AA. In total, these findings suggest that the structure of the metabolic network surrounding the generation and response to endogenous 2AA stress differs between S. enterica and E. coli. IMPORTANCE This work compared the metabolic networks surrounding the endogenous stressor 2-aminoacrylate in two closely related members of the Enterobacteriaceae. The data showed that despite the conservation of all relevant enzymes in this metabolic node, the two closely related organisms diverged in their metabolic network structures. This work highlights how a set of conserved components can generate distinct network architectures and how this can impact the physiology of an organism. This work defines a model to expand our understanding of the 2-aminoacrylate stress response and the differences in metabolic structures and cellular milieus between S. enterica and E. coli.

scholarly journals Non-random organization of flux control mechanisms in yeast central metabolic pathways

2021 ◽  
Author(s):  
Rosemary Yu ◽  
Egor Vorontsov ◽  
Carina Sihlbom ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Metabolic Network ◽  
Metabolic Pathways ◽  
Evolutionary History ◽  
Metabolic Flux ◽  
Metabolic Modeling ◽  
Control Mechanisms ◽  
Flux Control ◽  
Regulation Of Metabolism

Metabolic flux can be regulated by a variety of different mechanisms, but the organization of these mechanisms within the metabolic network has remained unknown. Here we test the hypothesis that flux control mechanisms are not distributed randomly in the metabolic network, but rather organized according to pathway. Combining proteomics, phosphoproteomics, and metabolic modeling, we report the largest collection of flux-enzyme-phosphoenzyme relationships to date in Saccharomyces cerevisiae. In support of the hypothesis, we show that (i) amino acid metabolic pathways are predominantly regulated by enzyme abundance stemming from transcriptional regulation; (ii) upper glycolysis and associated pathways, by inactivating enzyme phosphorylation; (iii) lower glycolysis and associated pathways, by activating enzyme phosphorylation; and (iv) glycolipid/glycophospholipid pathways, by a combination of enzyme phosphorylation and metabolic compartmentalization. We delineate the evolutionary history for the observed organization of flux control mechanisms in yeast central metabolic pathways, furthering our understanding of the regulation of metabolism and its evolution.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

scholarly journals Crossover Interference in Saccharomyces cerevisiae Requires a TID1/RDH54- and DMC1-Dependent Pathway

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1273-1286 ◽  
Author(s):  
Miki Shinohara ◽  
Kazuko Sakai ◽  
Akira Shinohara ◽  
Douglas K Bishop
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Tetrad Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crossover Interference ◽  
Meiotic Progression ◽  
Invasion Stage ◽  
Strand Invasion ◽  
Dependent Pathway

Abstract Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Δ mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Δ cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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