Regulation of arginine biosynthesis in Chlamydomonas reinhardii: Studies in vivo and of ornithine transcarbamoylase and argininosuccinate lyase activities

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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Regulation of Arginine Biosynthesis in the Psychropiezophilic Bacterium Moritella profunda: in vivo Repressibility and in vitro Repressor–Operator Contact Probing

Journal of Molecular Biology ◽

10.1016/s0022-2836(02)01375-x ◽

2003 ◽

Vol 326 (2) ◽

pp. 353-369 ◽

Cited By ~ 11

Author(s):

Ying Xu ◽

Yuan Sun ◽

Nadine Huysveld ◽

Daniel Gigot ◽

Nicolas Glansdorff ◽

...

Keyword(s):

Arginine Biosynthesis ◽

Moritella Profunda

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Metribuzin-Resistant Mutants of Chlamydomonas reinhardii

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-0526 ◽

1984 ◽

Vol 39 (5) ◽

pp. 437-439 ◽

Cited By ~ 17

Author(s):

N. Pucheu ◽

W. Oettmeier ◽

U. Heisterkamp ◽

K. Masson ◽

G.F. Wildner

Keyword(s):

Electron Transport ◽

Photosynthetic Electron Transport ◽

Wild Type ◽

Binding Studies ◽

Molecular Weight Range ◽

Chlamydomonas Reinhardii ◽

Thylakoid Membrane Proteins ◽

Mutant Strains ◽

Resistant Mutants

Herbicide resistance in Chlamydomonas reinhardii cells was induced by mutagenesis with 5-fluorodeoxyuridine and ethylmethanesulfonate. Four mutant strains were isolated and analyzed for resistance against DCMU-type or phenolic inhibitors of photosynthetic electron transport. The mutants were different in both the extent and the pattern of their resistance: the R/S value, i.e. the ratio of I50 values of the inhibition of photosynthetic electron transport in isolated resistant and susceptible thylakoids, varied for metribuzin from 10 000 to 36. The mutant MZ-1 was resistant against metribuzin, atrazine and DCMU, whereas the mutant MZ-2 showed resistance mainly against metribuzin and atrazine. The mutant MZ-3 was similar to MZ-1, but showed a lesser extent of resistance against DCMU. The mutant MZ-4 showed resistance against metribuzin, but not against atrazine. These results demonstrate that the resistance against one herbicide of the DCMU-type (metribuzin) must not be accompanied by similar resistance against te other inhibitors. Binding studies with radioactively labeled herbicides, [14C]metribuzin, [14C]atrazine and [3H]DCMU, and isolated thylakoids supported these observations. Phosphorylation of thylakoid membrane proteins was studied with wild-type cells and resistant mutants under in vivo conditions in the light. The 32P-labeled main proteins bands were in the molecular weight range of 10-14 kDa, 26-29 kDa, 32-35 kDa and 46-48 kDa. The pattern and the extent of incorporation of 32P were similar for the mutants and the wild-type cells.

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Temperature-sensitive breakdown in vivo of polysomes with mutants of Chlamydomonas reinhardii

MGG Molecular & General Genetics ◽

10.1007/bf00267568 ◽

1976 ◽

Vol 147 (2) ◽

pp. 169-177 ◽

Cited By ~ 1

Author(s):

John Cross ◽

Daniel McMahon

Keyword(s):

Temperature Sensitive ◽

Chlamydomonas Reinhardii

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Further studies of L-arginine biosynthesis in germinating pea seeds. Evidence for the presence of argininosuccinate synthetase in cotyledon extracts

Canadian Journal of Biochemistry ◽

10.1139/o69-073 ◽

1969 ◽

Vol 47 (4) ◽

pp. 467-475 ◽

Cited By ~ 6

Author(s):

P. D. Shargool ◽

E. A. Cossins

Keyword(s):

Enzyme Activity ◽

Gel Filtration ◽

Magnesium Ions ◽

Argininosuccinate Synthetase ◽

Argininosuccinate Lyase ◽

Specific Enzyme ◽

Arginine Biosynthesis ◽

Specific Enzyme Activity ◽

Pea Seeds

The synthesis and metabolism of arginine in germinating peas was examined by supplying micromolar quantities of L-citruiline-carbamyl-14C, DL-arginine-carbamyl-14C, and DL-arginine-5-14C to imbibing seeds. Citrulline was readily incorporated into arginine, but the labelled arginine solutions were not extensively metabolized.Extracts of 1-day-old cotyledons were found to catalyze the synthesis of arginine from citrulline in a reaction having absolute requirements for ATP, L-aspartate, and magnesium ions. The extracts were fractionated by (NH4)2SO4 precipitation followed by gel filtration on columns of Sephadex G-50 and G-200. These treatments increased the specific enzyme activity by approximately 36 times. After such treatments the preparations still contained appreciable amounts of argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) activity. The rate of arginine synthesis was altered by increasing the concentrations of L-citrulline, L-aspartate, and ATP. The latter compounds were found to be inhibitory at concentrations of 1 μmole/ml and 4 μmoles/ml, respectively. Arginine synthesis was markedly affected by pH and by additions of arginine and argininosuccinate. It is concluded that germinating pea cotyledons contain appreciable levels of argirrinosuccmate synthetase (L-citrulline:L-aspartate ligase (AMP), EC 6.3.4.5), and furthermore, that this enzyme has importance in arginine biosynthesis during germination.

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Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

Infection and Immunity ◽

10.1128/iai.70.6.3080-3084.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3080-3084 ◽

Cited By ~ 55

Author(s):

Bhavna G. Gordhan ◽

Debbie A. Smith ◽

Heidi Alderton ◽

Ruth A. McAdam ◽

Gregory J. Bancroft ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Auxotrophic Mutant ◽

Phenotypic Characterization ◽

Wild Type ◽

Arginine Biosynthesis ◽

High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

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Integrated Metabolic Profiling and Gene Expression Analysis Reveals Therapeutic Modalities in Breast Cancer

10.21203/rs.3.rs-117384/v1 ◽

2020 ◽

Author(s):

Chengheng Liao ◽

Cherise Ryan Glodowski ◽

Cheng Fan ◽

Juan Liu ◽

Kevin Raynard Mott ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Lines ◽

Metabolic Profiling ◽

Breast Cancer Cell Lines ◽

Breast Cancers ◽

Arginine Biosynthesis ◽

Metabolic Dysregulation

Abstract Metabolic dysregulation is one of the distinctive features in breast cancer. However, examining the metabolic features in various subtypes of breast cancer in their relationship to gene expression features in a physiologically relevant setting remains understudied. By performing metabolic profiling on triple-negative breast cancer (TNBC) and ER+ breast cancers from patients, TNBC patient-derived xenografts (PDXs), and representative breast cancer cell lines grown as tumors in vivo, we identify two distinctive groups defined by metabolites; a “Nucleotide-Enriched” group that shows high levels of pyrimidine pathway metabolites and biosynthetic enzymes, and a “Arginine Biosynthesis-Enriched” group that shows high levels of arginine biosynthesis intermediates. We reveal different metabolic enrichment profiles between cell lines grown in vitro versus in vivo, where cell lines grown in vivo more faithfually recapitulate patient tumors metabolic profiles. In addition, with integrated metabolic and gene expression profiling we identify a subset of genes that strongly correlates with the Nucleotide-Enriched metabolic profile, and which strongly predicts patient prognosis. As a proof-of-principle, when we target Nucleotide-Enriched metabolic dysregulation with a pyrimidine biosynthesis inhibitor (Brequinar), and/or a glutaminase inhibitor (CB-839), we observe therapeutic efficacy and decreased tumor growth in representative TNBC cell lines and an in vivo PDX upon combinatorial drug treatment. Our study reveals new therapeutic opportunities in breast cancer guided by a genomic biomarker, which could prove highly impactful for rapidly proliferating breast cancers specifically.

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Arginine synthesis by hepatomas in vitro. I. The requirements for cell growth in medium containing ornithine in place of arginine and the isolation and characterization of variant hepatomas auxotrophic for arginine

Journal of Cell Science ◽

10.1242/jcs.68.1.285 ◽

1984 ◽

Vol 68 (1) ◽

pp. 285-303 ◽

Cited By ~ 2

Author(s):

S.J. Goss

Keyword(s):

Cell Growth ◽

The Other ◽

Gene Products ◽

Carbamoyl Phosphate Synthetase ◽

Arginine Biosynthesis ◽

Carbamoyl Phosphate ◽

Isolation And Characterization ◽

Ornithine Transcarbamoylase

Cell growth in ‘ornithine-medium’ requires the expression of two liver-specific genes, those for ornithine transcarbamoylase (OTC) and carbamoyl phosphate synthetase I (CPS-I). CPS-II appears unable to replace CPS-I in this system. The need for N-acetylglutamate (to activate CPS-I) can be met, at least in part, by providing it in the medium. The other gene products involved in arginine biosynthesis are probably all ubiquitous (i.e. not tissue-specific). In an attempt to study the factors responsible for the expression of liver-specific genes, variant hepatomas are isolated that have lost the ability to grow in ornithine-medium. Two classes of ‘orn-’ variants are identified: unstable variants that require dexamethasone for adequate CPS-I production, and ‘stable’ variants that have lost many liver-specific traits. Studies on one stable variant show that it can revert (though rarely), and that it regains its various liver-specific traits in a non-coordinate fashion.

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In vivo-induced argininosuccinate lyase plays a role in the replication of Brucella abortus in RAW264.7 cells

Microbiology ◽

10.1099/mic.0.072926-0 ◽

2014 ◽

Vol 160 (3) ◽

pp. 567-575 ◽

Cited By ~ 2

Author(s):

Xiangan Han ◽

Xiaoqing Sun ◽

Xueqing Shan ◽

Min Zhang ◽

Jun Song ◽

...

Keyword(s):

Mutant Strain ◽

Brucella Abortus ◽

Raw264.7 Cells ◽

Mrna Levels ◽

Argininosuccinate Lyase ◽

Expression Library ◽

Genomic Expression ◽

Real Time Quantitative Pcr ◽

Positive Rate

Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308ΔASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308ΔASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308ΔASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P<0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.

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