scholarly journals EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

2021 ◽  
Vol 9 (3) ◽  
Author(s):  
Mario Alejandro Duque-Villegas ◽  
Bruno Lopes Abbadi ◽  
Paulo Ricardo Romero ◽  
Letícia Beatriz Matter ◽  
Luiza Galina ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Model Organism ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Building Blocks ◽  
Mycobacterial Species ◽  
Epsp Synthase ◽  
Content Type ◽  
Viable Cells ◽  
Starting Compound

We found that cells from Mycobacterium smegmatis , a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l- tryptophan, l -phenylalanine, and l -tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs.

scholarly journals A Mycobacterial Systems Resource for the Research Community

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
J. A. Judd ◽  
J. Canestrari ◽  
R. Clark ◽  
A. Joseph ◽  
P. Lapierre ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Protein Localization ◽  
Functional Characterization ◽  
Model Organism ◽  
Research Community ◽  
Mycobacterial Species ◽  
Biological Resource ◽  
Content Type ◽  
Core Genes

ABSTRACT Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis. To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis. This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulcerans. IMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

scholarly journals Evaluating aroA gene essentiality and EPSP synthase vulnerability in Mycobacterium smegmatis under different nutritional conditions

2020 ◽  
Author(s):  
Mario Alejandro Duque-Villegas ◽  
Bruno Lopes Abbadi ◽  
Paulo Ricardo Romero ◽  
Luiza Galina ◽  
Pedro Ferrari Dalberto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Smegmatis ◽  
Gene Knockout ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Gene Knockdown ◽  
Epsp Synthase ◽  
Gene Essentiality ◽  
Nutritional Conditions ◽  
Phosphate Synthase

AbstractThe epidemiological importance of bacteria from the genus Mycobacterium is indisputable and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important metabolites in bacteria, represents a target for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene knockout, gene knockdown and kinetic assays to evaluate aroA gene essentiality and the vulnerability of its protein product, EPSPS synthase from Mycobacterium smegmatis (MsEPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality of MsEPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions of aroA gene, together with kinetic assays using WT and mutant recombinant proteins, we show that aroA gene essentiality depends on MsEPSPS activity. To evaluate MsEPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces for aroA gene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating that MsEPSPS vulnerability depends on the environment conditions.ImportanceWe evaluated both gene essentiality and target vulnerability of the enzyme that catalyzes the sixth step of the shikimate pathway, the aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium smegmatis (MsEPSPS). Combining gene knockout experiments and kinetic assays, we established a causal link between aroA gene essentiality and the biological function of EPSPS protein, which we advocate is an indispensable step for target validation. Moreover, we characterized MsEPSPS vulnerability under different nutritional conditions and found it is a vulnerable target only when M. smegmatis is grown under poor nutritional conditions without supplementation with aromatic amino acids. Based on our findings, we suggest that gene essentiality information should be obtained from gene knockout experiments and not knockdown approaches, as even low levels of a protein after gene silencing can lead to a different growth phenotype when compared to that under its complete absence, as was the case with aroA and MsEPSPS in our study.

scholarly journals Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase

2020 ◽  
Author(s):  
Miia J. Rainio ◽  
Suvi Ruuskanen ◽  
Marjo Helander ◽  
Kari Saikkonen ◽  
Irma Saloniemi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Ecosystem Functions ◽  
Epsp Synthase

ABSTRACTGlyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (EPSPS) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in autotrophs. Our results reveal that bacteria easily become resistant to glyphosate through changes in the EPSPS active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

scholarly journals Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability

2019 ◽  
Vol 202 (5) ◽  
Author(s):  
Zdeněk Knejzlík ◽  
Klára Herkommerová ◽  
Dana Hocková ◽  
Iva Pichová
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Model Organism ◽  
Specific Inhibitor ◽  
Purine Salvage ◽  
Content Type ◽  
Acyclic Nucleoside Phosphonates ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis. Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed. IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5′-monophosphate from guanine and inosine-5′-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the Δhgprt M. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

scholarly journals SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions

Microbiology ◽  
2020 ◽  
Vol 166 (3) ◽  
pp. 296-305 ◽  
Author(s):  
Tsaone Tamuhla ◽  
Lydia Joubert ◽  
Danicke Willemse ◽  
Monique J. Williams
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Model Organism ◽  
Culture Conditions ◽  
Growth Defect ◽  
Content Type ◽  
Link Type ◽  
Targeted Gene Deletion ◽  
Accessory Factor

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

scholarly journals Enhanced Protocatechuic Acid Production From Glucose Using Pseudomonas putida 3-Dehydroshikimate Dehydratase Expressed in a Phenylalanine-Overproducing Mutant of Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Oliver Englund Örn ◽  
Stefano Sacchetto ◽  
Ed W. J. van Niel ◽  
Rajni Hatti-Kaul
Keyword(s):  
Escherichia Coli ◽  
Protocatechuic Acid ◽  
Feedback Inhibition ◽  
Critical Concentration ◽  
Membrane Damage ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Building Blocks ◽  
Muconic Acid ◽  
Batch Mode

Protocatechuic acid (PCA) is a strong antioxidant and is also a potential platform for polymer building blocks like vanillic acid, vanillin, muconic acid, and adipic acid. This report presents a study on PCA production from glucose via the shikimate pathway precursor 3-dehydroshikimate by heterologous expression of a gene encoding 3-dehydroshikimate dehydratase in Escherichia coli. The phenylalanine overproducing E. coli strain, engineered to relieve the allosteric inhibition of 3-deoxy-7-phosphoheptulonate synthase by the aromatic amino acids, was shown to give a higher yield of PCA than the unmodified strain under aerobic conditions. Highest PCA yield of 18 mol% per mol glucose and concentration of 4.2 g/L was obtained at a productivity of 0.079 g/L/h during cultivation in fed-batch mode using a feed of glucose and ammonium salt. Acetate was formed as a major side-product indicating a shift to catabolic metabolism as a result of feedback inhibition of the enzymes including 3-dehydroshikimate dehydratase by PCA when reaching a critical concentration. Indirect measurement of proton motive force by flow cytometry revealed no membrane damage of the cells by PCA, which was thus ruled out as a cause for affecting PCA formation.

scholarly journals Essentiality of Succinate Dehydrogenase in Mycobacterium smegmatis and Its Role in the Generation of the Membrane Potential Under Hypoxia

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Ildiko Pecsi ◽  
Kiel Hards ◽  
Nandula Ekanayaka ◽  
Michael Berney ◽  
Travis Hartman ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Succinate Dehydrogenase ◽  
Mycobacterium Smegmatis ◽  
Chemotherapeutic Agents ◽  
Differentially Expressed ◽  
Carbon Limitation ◽  
Mycobacterial Species ◽  
Content Type ◽  
Energy Limitation ◽  
The Individual

ABSTRACTSuccinate:quinone oxidoreductase (Sdh) is a membrane-bound complex that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putativesdhoperons, but the individual roles of these two operons are unknown. In this communication, we show thatMycobacterium smegmatismc2155 expresses two succinate dehydrogenases designated Sdh1 and Sdh2. Sdh1 is encoded by a five-gene operon (MSMEG_0416-MSMEG_0420), and Sdh2 is encoded by a four-gene operon (MSMEG_1672-MSMEG_1669). These two operons are differentially expressed in response to carbon limitation, hypoxia, and fumarate, as monitored bysdhpromoter-lacZfusions. While deletion of thesdh1operon did not yield any growth phenotypes on succinate or other nonfermentable carbon sources, thesdh2operon could be deleted only in a merodiploid background, demonstrating that Sdh2 is essential for growth. Sdh activity and succinate-dependent proton pumping were detected in cells grown aerobically, as well as under hypoxia. Fumarate reductase activity was absent under these conditions, indicating that neither Sdh1 nor Sdh2 could catalyze the reverse reaction. Sdh activity was inhibited by the Sdh inhibitor 3-nitroproprionate (3NP), and treatment with 3NP dissipated the membrane potential of wild-type or Δsdh1mutant cells under hypoxia but not that of cells grown aerobically. These data imply that Sdh2 is the generator of the membrane potential under hypoxia, an essential role for the cell.IMPORTANCEComplex II or succinate dehydrogenase (Sdh) is a major respiratory enzyme that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putativesdhoperons,sdh1andsdh2, but the individual roles of these two operons are unknown. In this communication, we show thatsdh1andsdh2are differentially expressed in response to energy limitation, oxygen tension, and alternative electron acceptor availability, suggesting distinct functional cellular roles. Sdh2 was essential for growth and generation of the membrane potential in hypoxic cells. Given the essentiality of succinate dehydrogenase and oxidative phosphorylation in the growth cycle ofMycobacterium tuberculosis, the potential exists to develop new antituberculosis agents against the mycobacterial succinate dehydrogenase. This enzyme has been proposed as a potential target for the development of new chemotherapeutic agents against intracellular parasites and mitochondrion-associated disease.

scholarly journals Development of anIn VitroAssay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria

2016 ◽  
Vol 60 (10) ◽  
pp. 6227-6233 ◽  
Author(s):  
Jessica Loraine ◽  
Feifei Pu ◽  
Obolbek Turapov ◽  
Galina V. Mukamolova
Keyword(s):  
Mycobacterium Smegmatis ◽  
Drug Exposure ◽  
Culture Supernatant ◽  
Model Organism ◽  
First Line ◽  
Drug Induced ◽  
Content Type ◽  
Resuscitation Promoting Factors ◽  
Major Infectious Disease ◽  
Significant Underestimation

ABSTRACTTuberculosis is a major infectious disease that requires prolonged chemotherapy with a combination of four drugs. Here we present data suggesting that treatment ofMycobacterium tuberculosis, the causative agent of tuberculosis, andMycobacterium smegmatis, a model organism widely used for the screening of antituberculosis agents, with first-line drugs resulted in the generation of substantial populations that could be recovered only by the addition of a culture supernatant from growing mycobacteria. These bacilli failed to grow in standard media, resulting in significant underestimation of the numbers of viable mycobacteria in treated samples. We generatedM. smegmatisstrains overexpressingM. tuberculosisresuscitation-promoting factors (Rpfs) and demonstrated their application for the detection of Rpf-dependent mycobacteria generated after drug exposure. Our data offer novel opportunities for validation of the sterilizing activity of antituberculosis agents.

scholarly journals Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogenAcinetobacter baumannii

2016 ◽  
Vol 72 (3) ◽  
pp. 179-187 ◽  
Author(s):  
Kristin A. Sutton ◽  
Jennifer Breen ◽  
Thomas A. Russo ◽  
L. Wayne Schultz ◽  
Timothy C. Umland
Keyword(s):  
Crystal Structure ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Epsp Synthase ◽  
Gene Encoding ◽  
Growth And Survival ◽  
Apicomplexan Parasites ◽  
Prephenate Dehydrogenase ◽  
Host Infection ◽  
Herbicide Glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphateviaa transferase reaction, and is the target of the herbicide glyphosate. TheAcinetobacter baumanniigene encoding EPSP synthase,aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctionalA. baumannii aroAgene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinantA. baumanniiEPSP (AbEPSP) synthase, comprising residues Ala301–Gln756 of thearoAgene product, was overexpressed inEscherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

scholarly journals Selective Requirement of the Shikimate Pathway of Legionella pneumophila for Intravacuolar Growth within Human Macrophages but Not within Acanthamoeba

2015 ◽  
Vol 83 (6) ◽  
pp. 2487-2495 ◽  
Author(s):  
Snake C. Jones ◽  
Christopher T. D. Price ◽  
Marina Santic ◽  
Yousef Abu Kwaik
Keyword(s):  
Amino Acids ◽  
Legionella Pneumophila ◽  
De Novo ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Human Monocyte ◽  
Optimal Growth ◽  
Growth Defect ◽  
Content Type ◽  
Human Macrophages

Legionella pneumophilautilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. AlthoughL. pneumophilautilizes host amino acids as the main sources of carbon and energy, it is not known whetherde novosynthesis of amino acids by intravacuolarL. pneumophilacontributes to its nutrition. ThearoBandaroEgenes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show thearoBandaroEmutants ofL. pneumophilato be defective in growth in human monocyte-derived macrophages (hMDMs) but not inAcanthamoebaspp. ThearoBandaroEmutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of thearoEbut not thearoBmutant. Therefore, the shikimate pathway ofL. pneumophilais differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within theAcanthamoebaspp. that synthesize the aromatic amino acids.

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