Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

ABSTRACT Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death. IMPORTANCE Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

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Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans

Eukaryotic Cell ◽

10.1128/ec.00373-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 656-666 ◽

Cited By ~ 9

Author(s):

Sandra L. Murray ◽

Michael J. Hynes

Keyword(s):

Aspergillus Nidulans ◽

Fluorescent Protein ◽

Citrate Synthase ◽

Glyoxylate Cycle ◽

Single Gene ◽

Carbon Sources ◽

Tca Cycle ◽

Poor Growth ◽

Content Type ◽

The Glyoxylate Cycle

ABSTRACT Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

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Genetics and Regulation of the Major Enzymes of Alanine Synthesis in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00738-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5304-5311 ◽

Cited By ~ 37

Author(s):

Sok Ho Kim ◽

Barbara L. Schneider ◽

Larry Reitzer

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Genetic Analysis ◽

Regulatory Protein ◽

Carbon Sources ◽

Optimal Growth ◽

Expression Studies ◽

Alanine Synthesis ◽

Multicopy Suppression

ABSTRACT Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with Km s for pyruvate within the intracellular range of pyruvate concentration.

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Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

mBio ◽

10.1128/mbio.00028-12 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 58

Author(s):

Max Chavarría ◽

Roelco J. Kleijn ◽

Uwe Sauer ◽

Katharina Pflüger-Grau ◽

Víctor de Lorenzo

Keyword(s):

Pseudomonas Putida ◽

Metabolic Networks ◽

Tca Cycle ◽

High Energy ◽

Pentose Phosphate ◽

Cell Physiology ◽

Phosphotransferase System ◽

Content Type ◽

Metabolic Fluxes ◽

Central Carbon

ABSTRACTTwo branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacteriumPseudomonas putidaKT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFruor PTSNtrgenes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtrprotein of the PTSNtrthus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology.IMPORTANCEThis study demonstrates that EIIANtrinfluences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars byP. putida. These findings indicate that the EIIANtrprotein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

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Salimesophilobacter vulgaris gen. nov., sp. nov., an anaerobic bacterium isolated from paper-mill wastewater

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.040915-0 ◽

2013 ◽

Vol 63 (Pt_4) ◽

pp. 1317-1322 ◽

Cited By ~ 14

Author(s):

Yan-Zhou Zhang ◽

Ming-Xu Fang ◽

Wen-Wu Zhang ◽

Tian-Tian Li ◽

Min Wu ◽

...

Keyword(s):

Type Species ◽

Sequence Similarity ◽

Heterotrophic Bacterium ◽

Carbon Sources ◽

Optimal Growth ◽

Paper Mill ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

Lateral Flagellum

A novel anaerobic, heterotrophic bacterium, designated strain Zn2T, was isolated from the wastewater of a paper mill in Zhejiang, China. Cells were Gram-type-positive rods, 0.5–0.8 µm wide and 2–4 µm long, and were motile by a lateral flagellum. The ranges of temperature and pH for growth were 10–50 °C and pH 6.0–9.5. Optimal growth occurred at 35 °C and pH 7.3–7.5. The strain did not require NaCl for growth, but its inclusion in the medium improved growth (optimum concentration 6 %). Substrates utilized as sole carbon sources were peptone, tryptone, Casamino acids, d-xylose, salicin, glycerol, formate, acetate and propionate. The main products of carbohydrate fermentation were acetate, formate, propionate and lactate. Elemental sulfur, thiosulfate and Fe(III) were used as electron acceptors, but sulfate, sulfite, nitrate, nitrite and Mn(IV) were not. Growth was inhibited by the addition of 10 µg ampicillin, penicillin, tetracycline or chloramphenicol ml−1. iso-C15 : 0, C14 : 0, C16 : 0, C16 : 1 cis9 and C18 : 1 cis9 were the major fatty acids. Strain Zn2T did not contain any detectable menaquinones or ubiquinones. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, two unknown phospholipids and four unknown glycolipids. The genomic DNA G+C content was 37 mol%, as determined by HPLC. 16S rRNA gene sequence analysis revealed that strain Zn2T was a member of family Clostridiaceae , and was most closely related to the type strains of Geosporobacter subterraneus , Thermotalea metallivorans and Caminicella sporogenes , showing 91.2, 90.3 and 91.1 % sequence similarity, respectively. On the basis of its phenotypic and genotypic properties, strain Zn2T is suggested to represent a novel species of a new genus, for which the name Salimesophilobacter vulgaris gen. nov., sp. nov. is proposed. The type strain of Salimesophilobacter vulgaris is Zn2T ( = DSM 24770T = JCM 17796T).

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NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2Fixation Capabilities

Applied and Environmental Microbiology ◽

10.1128/aem.06412-11 ◽

2012 ◽

Vol 78 (8) ◽

pp. 2803-2812 ◽

Cited By ~ 13

Author(s):

Ye Zhang ◽

Toshihiro Aono ◽

Phillip Poole ◽

Turlough M. Finan

Keyword(s):

Malic Enzyme ◽

Sinorhizobium Meliloti ◽

Phosphoenolpyruvate Carboxykinase ◽

Rhizobium Leguminosarum ◽

Dicarboxylic Acids ◽

Dry Weight ◽

Tca Cycle ◽

Broad Host Range ◽

Azorhizobium Caulinodans ◽

Content Type

ABSTRACTC4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. InSinorhizobium melilotibacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiontRhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report thatdmemutants of the broad-host-rangeSinorhizobiumsp. strain NGR234 formed nodules whose level of N2fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while singlepckAand singledmemutants fixed N2at reduced rates, apckA dmedouble mutant had no N2-fixing activity (Fix−). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix−phenotype ofS. meliloti dmemutants may be specific to the alfalfa-S. melilotisymbiosis. We therefore examined the ME-like genesazc3656andazc0119fromAzorhizobium caulinodans, asazc3656mutants were previously shown to form Fix−nodules on the tropical legumeSesbania rostrata. We found that purified AZC3656 protein is an NAD(P)+-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N2fixation inA. caulinodansandS. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

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Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

Journal of Bacteriology ◽

10.1128/jb.02333-14 ◽

2014 ◽

Vol 197 (4) ◽

pp. 749-761 ◽

Cited By ~ 33

Author(s):

M. A. Serbanescu ◽

M. Cordova ◽

K. Krastel ◽

R. Flick ◽

N. Beloglazova ◽

...

Keyword(s):

Heat Shock ◽

Streptococcus Mutans ◽

Antibiotic Resistance Genes ◽

Transcriptional Analysis ◽

Cell Physiology ◽

Type I ◽

Type Ii ◽

Content Type ◽

Bacterial Physiology

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

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Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01400-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5960-5968 ◽

Cited By ~ 5

Author(s):

Tomohiro Shimada ◽

Kan Tanaka

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Monitoring System ◽

Carbon Sources ◽

Tca Cycle ◽

Bacterial Cells ◽

Bacterial Luciferase ◽

Content Type ◽

Time Dynamics ◽

The Tca Cycle

ABSTRACTRegulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employingVibrio fischeriluciferase to probe the intracellular metabolic condition inEscherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used aVibrio fischeriluciferase monitoring system to probe the intracellular metabolic condition inEscherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.

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Description of Thermogemmatispora carboxidivorans sp. nov., a carbon-monoxide-oxidizing member of the class Ktedonobacteria isolated from a geothermally heated biofilm, and analysis of carbon monoxide oxidation by members of the class Ktedonobacteria

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.059675-0 ◽

2014 ◽

Vol 64 (Pt_4) ◽

pp. 1244-1251 ◽

Cited By ~ 58

Author(s):

C. E. King ◽

G. M. King

Keyword(s):

Carbon Monoxide ◽

Type Species ◽

Phylogenetic Analyses ◽

Carbon Sources ◽

Optimal Growth ◽

Rrna Gene ◽

Biochemical Tests ◽

Orange Pigment ◽

Content Type ◽

Link Type

A thermophilic, aerobic, Gram-stain-positive bacterium (strain PM5T), which formed mycelia of irregularly branched filaments and produced multiple exospores per cell, was isolated from a geothermally heated biofilm. Strain PM5T grew at 40–65 °C and pH 4.1–8.0, with optimal growth at 55 °C and pH 6.0. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain PM5T belonged to the class Ktedonobacteria , and was related most closely to Thermogemmatispora onikobensis ONI-1T (97.7 % similarity) and Thermogemmatispora foliorum ONI-5T (96.1 %). Morphological features and fatty acid profiles (major fatty acids: iso-C17 : 0, iso-C19 : 0 and 12,17-dimethyl C18 : 0) supported the affiliation of strain PM5T to the genus Thermogemmatispora . Strain PM5T oxidized carbon monoxide [CO; 10±1 nmol h−1 (mg protein)−1], but did not grow with CO as a sole carbon and energy source. Results from analyses of related strains indicated that the capacity for CO uptake occurred commonly among the members of the class Ktedonobacteria ; 13 of 14 strains tested consumed CO or harboured coxL genes that potentially enabled CO oxidation. The results of DNA–DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain PM5T from the two recognized species of the genus Thermogemmatispora . Strain PM5T differed from Thermogemmatispora onikobensis ONI-1T in its production of orange pigment, lower temperature optimum, hydrolysis of casein and starch, inability to grow with mannitol, xylose or rhamnose as sole carbon sources, and utilization of organic acids and amino acids. Strain PM5T is therefore considered to represent a novel species, for which the name Thermogemmatispora carboxidivorans sp. nov. is proposed. The type strain is PM5T ( = DSM 45816T = ATCC BAA-2534T).

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Two Pathways for Glutamate Biosynthesis in the Syntrophic Bacterium Syntrophus aciditrophicus

Applied and Environmental Microbiology ◽

10.1128/aem.02323-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8434-8444 ◽

Cited By ~ 5

Author(s):

Marie Kim ◽

Huynh M. Le ◽

Xiulan Xie ◽

Xueyang Feng ◽

Yinjie J. Tang ◽

...

Keyword(s):

Amino Acids ◽

Citrate Synthase ◽

Carbon Sources ◽

Krebs Cycle ◽

Content Type ◽

Cell Extracts ◽

Cyclohexane Carboxylate ◽

Glutamate Synthesis ◽

Glutamate Biosynthesis ◽

Syntrophus Aciditrophicus

ABSTRACTThe anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate bySyntrophus aciditrophicusgrown syntrophically withMethanospirillum hungateiprovides a model to study syntrophic cooperation. Recent studies revealed thatS. aciditrophicuscontainsRe-citrate synthase but lacks the commonSi-citrate synthase. To establish whether theRe-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-13C]acetate and [1-14C]acetate as well as [13C]bicarbonate as additional carbon sources during axenic growth ofS. aciditrophicuson crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and13C nuclear magnetic resonance (NMR) spectroscopy of the obtained [13C]glutamate, as well as decarboxylation of [14C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route usedRe-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium ofS. aciditrophicuswhen grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. BesidesClostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.

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Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis

10.1101/2021.06.21.449354 ◽

2021 ◽

Author(s):

Cara R Adolph ◽

Matthew B McNeil ◽

Gregory M. Cook

Keyword(s):

Mycobacterium Tuberculosis ◽

Respiratory Chain ◽

Focal Point ◽

Single Gene ◽

Carbon Sources ◽

Optimal Growth ◽

Tca Cycle ◽

Drug Susceptibility ◽

Succinate Oxidation ◽

Antitubercular Drugs

Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the TCA cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bi-directional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 + sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 + sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of several antitubercular drugs against M. tuberculosis, including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.

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