scholarly journals Insertion of Transposon Tn5tac1 in the Sinorhizobium meliloti Malate Dehydrogenase (mdh) Gene Results in Conditional Polar Effects on Downstream TCA Cycle Genes

2004 ◽  
Vol 17 (12) ◽  
pp. 1318-1327 ◽  
Author(s):  
Sergiy l. Dymov ◽  
David J. J. Meek ◽  
Blaire Steven ◽  
Brian T. Driscoll
Keyword(s):  
Malate Dehydrogenase ◽  
Sinorhizobium Meliloti ◽  
Tricarboxylic Acid Cycle ◽  
Root Nodules ◽  
Carbon Sources ◽  
Dry Weight ◽  
Tca Cycle ◽  
Growth Conditions ◽  
Oxoglutarate Dehydrogenase ◽  
Succinyl Coa Synthetase

To isolate Sinorhizobium meliloti mutants deficient in malate dehydrogenase (MDH) activity, random transposon Tn5tac1 insertion mutants were screened for conditional lethal phenotypes on complex medium. Tn5tac1 has an outward-oriented isopropyl-β-D-thiogalactopyranoside (IPTG)- inducible promoter (Ptac). The insertion in strain Rm30049 was mapped to the mdh gene, which was found to lie directly upstream of the genes encoding succinyl-CoA synthetase (sucCD) and 2-oxoglutarate dehydrogenase (sucAB and lpdA). Rm30049 required IPTG for wild-type growth in complex media, and had a complex growth phenotype in minimal media with different carbon sources. The mdh∷ Tn5tac1 insertion eliminated MDH activity under all growth conditions, and activities of succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase were affected by the addition of IPTG. Reverse-transcriptase polymerase chain reaction (RT-PCR) studies confirmed that expression from Ptac was induced by IPTG and leaky in its absence. Alfalfa plants inoculated with Rm30049 were chlorotic and stunted, with small white root nodules, and had shoot dry weight and percent-N content values similar to those of uninoculated plants. Cosmid clone pDS15 restored MDH activity to Rm30049, complemented both the mutant growth and symbiotic phenotypes, and was found to carry six complete (sdhB, mdh, sucCDAB) and two partial (lpdA, sdhA) tricarboxylic acid cycle genes.

scholarly journals Disruption of the MreB Elongasome Is Overcome by Mutations in the Tricarboxylic Acid Cycle

2021 ◽  
Vol 12 ◽  
Author(s):  
Brody Barton ◽  
Addison Grinnell ◽  
Randy M. Morgenstein
Keyword(s):  
Cell Wall ◽  
Malate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Normal Growth ◽  
Tca Cycle ◽  
Growth Conditions ◽  
Tricarboxylic Acid ◽  
Uridine Diphosphate ◽  
Metabolite Analysis ◽  
The Media

The bacterial actin homolog, MreB, is highly conserved among rod-shaped bacteria and essential for growth under normal growth conditions. MreB directs the localization of cell wall synthesis and loss of MreB results in round cells and death. Using the MreB depolymerizing drug, A22, we show that changes to central metabolism through deletion of malate dehydrogenase from the tricarboxylic acid (TCA) cycle results in cells with an increased tolerance to A22. We hypothesize that deletion of malate dehydrogenase leads to the upregulation of gluconeogenesis resulting in an increase in cell wall precursors. Consistent with this idea, metabolite analysis revealed that malate dehydrogenase (mdh) deletion cells possess elevated levels of several glycolysis/gluconeogenesis compounds and the cell wall precursor, uridine diphosphate N-acetylglucosamine (UDP-NAG). In agreement with these results, the increased A22 resistance phenotype can be recapitulated through the addition of glucose to the media. Finally, we show that this increase in antibiotic tolerance is not specific to A22 but also applies to the cell wall-targeting antibiotic, mecillinam.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

Effect of thermal injury on the TCA cycle enzymes of Staphylococcus aureus MF 31 and Salmonella typhimurium 7136

1971 ◽  
Vol 17 (6) ◽  
pp. 759-765 ◽  
Author(s):  
Richard I. Tomlins ◽  
Merle D. Pierson ◽  
Z. John Ordal
Keyword(s):  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Thermal Injury ◽  
Specific Activity ◽  
Fumarate Hydratase ◽  
Tca Cycle ◽  
Tca Cycle Enzymes ◽  
The Tca Cycle ◽  
Oxoglutarate Dehydrogenase ◽  
Metabolic Activities

The heating of S. aureus MF-31 and S. typhimurium 7136 at 52C and 48C respectively, produced a sublethal heat injury. When injured cells were placed in fresh growth medium they recovered. The recovery of S. aureus was not inhibited by chloramphenicol. The metabolic activities of tricarboxylic acid (TCA) cycle enzymes, as well as other selected enzymes in crude extracts of normal and heat-injured cells of both microorganisms were assayed. In extracts from S. typhimurium there was some loss of specific activity with fumarate hydratase, glutamate dehydrogenase, fructose diphosphate aldolase, lactate dehydrogenase, and the NAD(P) oxidases as a result of heating. In extracts from S. aureus oxoglutarate dehydrogenase, malate dehydrogenase and lactate dehydrogenase were severely inactivated after heating. Other enzymes in comparison were only moderately sensitive to heat. No significant increase in enzyme activity was observed in extracts from injured cells of either microorganism. Re-naturation of lactate dehydrogenase and malate dehydrogenase occurred during the recovery of S. aureus both in the presence and absence of chloramphenicol. No renaturation of oxoglutarate dehydrogenase was found under the same conditions.

The assimilation of bicarbonate by Neocosmospora vasinfecta

1969 ◽  
Vol 15 (5) ◽  
pp. 389-398 ◽  
Author(s):  
K. Budd
Keyword(s):  
Protein Synthesis ◽  
Carbon Source ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Dry Weight ◽  
Insoluble Fraction ◽  
Tricarboxylic Acid ◽  
End Products ◽  
Almost All ◽  
Exogenous Nitrogen

The assimilation of 14C-bicarbonate under controlled conditions was examined in midlog-phase mycelium grown on dextrose as sole carbon source. Sustained assimilation depended on the presence of exogenous nitrogen and carbon sources. When these were provided, assimilation rates of 20–30 μmoles/hour per 100 mg dry weight were maintained for at least 4 hours. After the second hour, almost all of the assimilated bicarbonate-C entered the 80% ethanol-insoluble fraction. Amino acids, especially aspartic and glutamic, were the main destination of assimilated bicarbonate-C; nucleic acids and acids of the tricarboxylic acid cycle accounted for smaller amounts of this carbon. The apparent Km for overall assimilation was 1.4 – 2.2 × 10−4 M with respect to bicarbonate.Assimilation was inhibited by inhibitors of protein synthesis, especially actidione and p-fluorophenylalanine. Evidence was obtained for regulation of assimilation by its end products, and also by the carbon source on which the mycelium was grown. It is concluded that assimilation of bicarbonate or CO2 has an anaplerotic function during protein synthesis in this organism.

scholarly journals Isolation and Molecular Identification of Phenol Degrading Bacterium from Industrial Wastes, Collected from Jeddah Saudi Arabia

2021 ◽  
Vol 14 (4) ◽  
pp. 1992-2001
Author(s):  
Bothaina A. Alaidaroos
Keyword(s):  
Phenol Degradation ◽  
Carbon Sources ◽  
Dry Weight ◽  
Degradation Process ◽  
Industrial Area ◽  
Growth Conditions ◽  
Industrial Wastes ◽  
Aquatic Life ◽  
Environment And Health ◽  
Degrading Bacterium

In the past two decades, phenolic compounds have had different applications, however their use in densification has increased considerably due to Covid 19. Discharge of these dangerous materials is highly toxic and causes risk and severe problems to the environment and health of human and animals, in addition to it being harmful to the aquatic life. Phenol degradation is very important due to high toxicity and stability. The aim of this study is to isolate phenol-degrading aerobic bacteria from hydrocarbon contaminated soil or wastewater, collected from the industrial area of Jeddah. Minimal medium containing phenol as carbon source was used to isolate different bacteria. About 30 actinomycete isolates were obtained, purified and preserved on Starch nitrate. Out of 30 isolates, eight isolates (27%) grow well in medium containing 0.1% phenol. After growing in broth medium, isolate BA4 and isolate BA8 were very active in phenol degradation. Growth and phenol degradation was measured in liquid medium for the two isolates. Morphological and physiological characters of these isolates were detected using different methods. Using molecular methods, they were belonging to a genus of actinomycetes. They were identified as Streptomyces flavabus BA4 and Streptomyces sp. BA8.The effects of some growth factors on growth and phenol degradation were determined. Growth was measured by dry weight (mg/l) while phenol degradation was detected by assaying the residual phenol concentration. The presence of electron donors such as glucose, starch, glycine, peptone, and Na acetate affect both growth and phenol degradation. It was clear that addition of 1 g/l peptone enhanced both growth and phenol degradation. The isolate use phenol and its derivatives m-cresol and o-cresol as carbon sources and addition of vitamin B complex increased the bacterial growth. In conclusion, phenol degradation was detected by actinobacteria and was affected by some physical and biochemical factors. It was noticed that optimization of growth conditions enhanced both growth and phenol degradation by the two selected Streptomyces isolate. Degradation process by isolate BA4 could be a promising solution for removal of phenol from wastewater.

Precision Cultivation of Phaeodactylum tricornutum and Free Lipid Acid Analysis

2011 ◽  
Vol 354-355 ◽  
pp. 226-230
Author(s):  
Feng He ◽  
Peng Cheng Fu ◽  
Chun Ming Xu
Keyword(s):  
Fatty Acid ◽  
Phaeodactylum Tricornutum ◽  
Cell Concentration ◽  
Fatty Acid Content ◽  
Carbon Sources ◽  
Total Lipid Content ◽  
Biomass Accumulation ◽  
Dry Weight ◽  
Optimal Growth ◽  
Growth Conditions

Abstract：A field test was conducted on a photobioreactors for cultivaion of P. tricornutum for optimization cultivation conditions.Here,we use a flat-cuvette pohotobioreactor to control the irradiance, pH, gas composition combined with on-line monitoring by fluorometer and densitometer.By chosing the culture medium in containing glucose，sodium acetate and glycerol, to check for the biomass, cell concentration, biochemical substances and fatty acid content nearly three months . The results show that: Phaeodactylum tricornutum is not only photoautotroph but also mixotroph, which with selectivity for substance concentration and organic carbon sources, the optimum concentration of glucose is 20mmol/L, the optimal growth condition in 500mL flask contains that the temperature is 25±1°C, the light intensity is 50µmol/m2.s, the pH is 8.5. Whatever the carbon sources are able to promote the biomass accumulation. When the cell concentration achieve to 2.5×106 cells/mL in the end of the culture, analyzed total lipid content so as to determine the biomass accumulation and biomass variation in different growth conditions. By ultrasonic extracting and freeze drying, the total of lipid reaches 20%(dry weight); the main content of fatty acid is C16:0,C18:0,which is the better material for biodisel production.

scholarly journals Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of Salmonella enterica Serovar Typhimurium in BALB/c Mice

2006 ◽  
Vol 74 (2) ◽  
pp. 1130-1140 ◽  
Author(s):  
Merlin Tchawa Yimga ◽  
Mary P. Leatham ◽  
James H. Allen ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Glyoxylate Bypass ◽  
The Tca Cycle ◽  
Mouse Tissues ◽  
Serovar Typhimurium ◽  
Genes Encoding

ABSTRACT In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δcra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ΔsdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ΔaspA mutant and an SR-11 ΔfrdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ΔfadD mutant and an SR-11 ΔaceA mutant were both fully virulent.

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

2012 ◽  
Vol 78 (8) ◽  
pp. 2803-2812 ◽  
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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