scholarly journals Thiamine Is Synthesized by a Salvage Pathway in Rhizobium leguminosarum bv. viciae Strain 3841

2006 ◽  
Vol 188 (18) ◽  
pp. 6661-6668 ◽  
Author(s):  
R. Karunakaran ◽  
K. Ebert ◽  
S. Harvey ◽  
M. E. Leonard ◽  
V. Ramachandran ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
De Novo ◽  
Fluorescent Microscopy ◽  
Good Growth ◽  
Wild Type ◽  
Salvage Pathway ◽  
Mesorhizobium Loti ◽  
Reverse Transcription Pcr

ABSTRACT In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only “pin” colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(β-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

scholarly journals Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Michael J. Mitsch ◽  
George C. diCenzo ◽  
Alison Cowie ◽  
Turlough M. Finan
Keyword(s):  
Nitrogen Fixation ◽  
Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Changes ◽  
Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

scholarly journals Glutathione Status in the Roots of Tomato Plants Transgenic by Genes psl and rapA1 in the Presence of Rhizobium leguminosarum

2021 ◽  
Vol 68 (5) ◽  
pp. 923-930
Author(s):  
Z. R. Vershinina ◽  
O. V. Chubukova ◽  
D. R. Maslennikova
Keyword(s):  
Solanum Lycopersicum ◽  
Bacterial Adhesion ◽  
Rhizobium Leguminosarum ◽  
De Novo ◽  
Growth Parameters ◽  
Wild Type ◽  
Leguminous Plants ◽  
Tomato Plants ◽  
Glutathione Status

Abstract The level of glutathione was investigated in the roots of tomato (Solanum lycopersicum L.) plants transgenic by genes psl and rapA1 in the presence of a microsymbiont of leguminous plants Rhizobium leguminosarum VSy3. The plants transformed with gene psl showed a greater bacterial adhesion than the plants transformed with gene rapA1, which positively correlated with growth parameters of plants. Treatment with rhizobia elevated the content of glutathione in the roots of wild type plants three times, 4.7 times in the roots of plants transformed with gene rapA1, and more than five times in the plants transgenic by gene psl. The obtained results suggest that the level of glutathione in the roots may serve as a marker of efficiency of artificial symbiotic systems produced de novo.

scholarly journals Regulation and characterization of mutants of fixABCX in Rhizobium leguminosarum.

2021 ◽  
Author(s):  
Isabel Webb ◽  
Jiabao Xu ◽  
Carmen Sanchez-Cañizares ◽  
Ramakrishnan Karunakaran ◽  
Vinoy Ramachandran ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Rhizobium Leguminosarum ◽  
Redox Balance ◽  
Rna Seq ◽  
Wild Type ◽  
Transcription Start Sites ◽  
Sym Plasmid ◽  
Microaerobic Conditions ◽  
Type Infection

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase, whilst still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, electron transfer flavoproteins essential for nitrogen fixation, are encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically-regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5’-end mapping of transcription start sites using differential (d) RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.

scholarly journals Antioxidant ability of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841

2020 ◽  
Author(s):  
Qian Zou ◽  
Yanlin Zhou ◽  
Guojun Cheng ◽  
Yang Peng ◽  
Sha Luo ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Quantitative Proteomics ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Proteome Analysis ◽  
Wild Type ◽  
Triple Mutant ◽  
Antioxidant Proteins ◽  
Mixed Disulfides ◽  
Nodule Symbiosis

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum bv. Viciae 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grxB mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grxB mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis. IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

scholarly journals Binding Site Determinants for the LysR-Type Transcriptional Regulator PcaQ in the Legume Endosymbiont Sinorhizobium meliloti

2007 ◽  
Vol 190 (4) ◽  
pp. 1237-1246 ◽  
Author(s):  
Allyson M. MacLean ◽  
Michelle I. Anstey ◽  
Turlough M. Finan
Keyword(s):  
Binding Site ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Transcriptional Start Site ◽  
Mesorhizobium Loti ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

ABSTRACT LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the β-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions −78 to −45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many α-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

Pathways for phosphatidylcholine biosynthesis in bacteria

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3461-3471 ◽  
Author(s):  
Fernando Martínez-Morales ◽  
Max Schobert ◽  
Isabel M. López-Lara ◽  
Otto Geiger
Keyword(s):  
Borrelia Burgdorferi ◽  
Legionella Pneumophila ◽  
Bradyrhizobium Japonicum ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Brucella Melitensis ◽  
Membrane Lipid ◽  
Mesorhizobium Loti ◽  
Methylation Pathway ◽  
Phosphatidylcholine Biosynthesis

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

Mechanism of pyridoxine 5'-phosphate accumulation in PLPBP protein-deficiency

2022 ◽  
Author(s):  
Tomokazu Ito ◽  
Honoka Ogawa ◽  
Hisashi Hemmi ◽  
Diana M. Downs ◽  
Tohru Yoshimura
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Vitamin B ◽  
Wild Type ◽  
Salvage Pathway ◽  
Intracellular Accumulation ◽  
Pyridoxal Kinase ◽  
Genetic Studies ◽  
E Coli ◽  
Metabolic Systems

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B 6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B 6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. The accumulated PNP could affect diverse metabolic systems through inhibition of some PLP-dependent enzymes. In this study, we investigated the as yet unclear mechanism of intracellular accumulation of PNP by the loss of PLPBP protein encoded by yggS in E. coli . Genetic studies using several PLPBP-deficient strains of E. coli lacking known enzyme(s) in the de novo or salvage pathway of vitamin B 6 , which includes pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies with the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa . We show that disruption of PLPBP lead to perturb PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. Importance A PLP-binding protein PLPBP from the conserved COG0325 family has recently been recognized as a key player in vitamin B 6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B 6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of the PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of the PNP accumulation was poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and its disruption may lead to the accumulation of PNP in PLPBP-deficiency.

scholarly journals Nickel Availability and hupSL Activation by Heterologous Regulators Limit Symbiotic Expression of theRhizobium leguminosarum bv. Viciae Hydrogenase System in Hup− Rhizobia

2000 ◽  
Vol 66 (3) ◽  
pp. 937-942 ◽  
Author(s):  
Belén Brito ◽  
Jorge Monza ◽  
Juan Imperial ◽  
Tomás Ruiz-Argüeso ◽  
Jose Manuel Palacios
Keyword(s):  
Transcriptional Activation ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Transcriptional Regulator ◽  
Hydrogen Uptake ◽  
Hydrogenase Activity ◽  
Structural Genes ◽  
Mesorhizobium Loti ◽  
Genes Expression ◽  
Relevant Factors

ABSTRACT A limited number of Rhizobium andBradyrhizobium strains possess a hydrogen uptake (Hup) system that recycles the hydrogen released from the nitrogen fixation process in legume nodules. To extend this ability to rhizobia that nodulate agronomically important crops, we investigated factors that affect the expression of a cosmid-borne Hup system from Rhizobium leguminosarum bv. viciae UPM791 in R. leguminosarumbv. viciae, Rhizobium etli, Mesorhizobium loti, and Sinorhizobium meliloti Hup− strains. After cosmid pAL618 carrying the entire hup system of strain UPM791 was introduced, all recipient strains acquired the ability to oxidize H2 in symbioses with their hosts, although the levels of hydrogenase activity were found to be strain and species dependent. The levels of hydrogenase activity were correlated with the levels of nickel-dependent processing of the hydrogenase structural polypeptides and with transcription of structural genes. Expression of the NifA-dependent hupSL promoter varied depending on the genetic background, while the hyp operon, which is controlled by the FnrN transcriptional regulator, was expressed at similar levels in all recipient strains. With the exception of theR. etli-bean symbiosis, the availability of nickel to bacteroids strongly affected hydrogenase processing and activity in the systems tested. Our results indicate that efficient transcriptional activation by heterologous regulators and processing of the hydrogenase as a function of the availability of nickel to the bacteroid are relevant factors that affect hydrogenase expression in heterologous rhizobia.

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