Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.     

Germination of Bacillus cereus spores induced by purine ribosides and their analogs: effects of modification of base and sugar moieties of purine nucleosides on germination-inducing activity

Purine riboside and some of its analogs were tested for their ability to induce germination of Bacillus cereus T spores. Hypoxanthine and adenine showed no germination-inducing activity either in the present or absence of D-ribose or its phosphorylated derivatives. Purine riboside and 18 analogs with modified purine base were all able to induce germination of the spores to various extents. In contrast to this, the requirement for the sugar moiety in the purine riboside appeared to be more stringent. Only those nucleosides that contained either D-ribose or deoxy-D-ribose, and certain species of azole derivatives such as 5-aminoimidazole-4-carboxamide covalently linked to the C(1') of the sugar actively induced germination.     

Decreased purine synthesis during amino acid starvation of human lymphoblasts

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.     

Rhizobium meliloti purine auxotrophs arenod + but defective in nitrogen fixation

Several purine auxotrophs were isolated inRhizobium meliloti and characterized for their nutritional requirements. They were found to produce small, irregular nodules lacking any detectable nitrogenase activity onMedicago sativa. The symbiotic aberration manifests itself only in the late developmental stage, for, (i) these purine auxotrophs infect theMedicago sativa root hairs by forming normal infection threads, and (ii) the mutants are recovered from the root nodules induced by them. External supplementation of the plant growth substrate with purines or their biosynthetic intermediates fails to restore symbiosis. This, and the failure of complementation of these auxotrophs with the known symbiotic genes, demonstrates that these mutants perhaps define a new set of genes influencing the symbiotic process inRhizobium meliloti.     

The role of histidine in regulating the synthesis of purine nucleotides in Escherichia coli cells]

Coordination of GTP and 5-aminoimidazole-4-carboxamide riboside 5'-phosphate pools changes was studied. The CTP pool is an important component of Escherichia coli metabolism, while AICAR 5'-phosphate being one of alarmones controls the synthesis of GTP. Main attention was paid to histidine, the biosynthesis of which is connected with formation of purine nucleotides. The expression of the histidine operon and biosynthesis of histidine are shown to change the AICAR pool and help the formation of the GTP pool. The ribosomal antibiotics streptomycin and chloramphenicol may cause the temporary deficiency of GTP eliminated by the increase of alarmone AICAR pool. The latter event is concluded to cause the increase in GTP pool independent of the means of AICAR accumulation (C1-pholatedependent restriction of metabolization or, vice versa, the stimulation in the histidine biosynthesis pathway).     

Pleiotropic effects of purine auxotrophy inRhizobium meliloti on cell surface molecules

Rhizobial purine auxotrophs have earlier been shown to be defective in symbiosis, though the exact reason for this failure is not clear. Using various dyes that specifically bind different cell surface molecules, we show that there are multiple changes in the cell surface molecules associated with different purine auxotrophs. Affected molecules in different purine auxotrophs that were tested include (i) acidic exopolysaccharides, (ii) cellulose fibrils, and (iii) beta (1–3) glucans. Our results show that the symbiotic deficiency of purine auxotrophs is likely to be a result of these associated changes on the cell surface     

Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10⁵ to 10⁶ bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.     

Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates

1. The pattern of distribution on the purine pathway of mutants of Salmonella typhimurium LT2 that had the double growth requirement for a purine plus the pyrimidine moiety of thiamine (ath mutants) indicated that purines and the pyrimidine moiety of thiamine share the early part of their biosynthetic pathways, and that 4-aminoimidazole ribonucleotide (AIR) is the last common intermediate. Two mutants that at first appeared anomalous were further investigated and found not to affect this deduction. 2. The ribonucleoside form of AIR (AIR(s)) satisfied the requirements both for a purine and for the pyrimidine moiety of thiamine of an ath mutant. 3. Methionine was required for the conversion of AIR into the pyrimidine moiety. 4. Radioactive AIR(s) was converted into radioactive pyrimidine moiety by an ath mutant without significant dilution of specific radioactivity. 5. Possible mechanisms for pyrimidine-moiety biosynthesis from AIR are discussed.     

Chromatographic analysis of purine precursors in mouse L1210 leukemia

A number of antagonists of nucleotide metabolism with anti-cancer activity affect the de novo purine pathway. To determine the biochemical mechanisms of cytotoxicity of these drugs, assay procedures have been developed for measurement of the levels of intermediates proximal to IMP in the pathway for de novo purine biosynthesis in mouse L1210 leukemia cells. Purine precursors have been synthesized in vitro from [14C]glycine using enzymes from chicken liver. These 14C-labeled intermediates have been used as marker compounds to define retention times for metabolites of leukemia cells separated by HPLC and the chromatographic mobilities of these intermediates after two-dimensional thin-layer chromatography. These new chromatographic procedures have been used in combination to determine the steady-state concentrations for purine precursors in mouse L1210 leukemia cells in the exponential phase of growth: N-formylglycineamide ribotide (16 microM); N-formylglycineamidine ribotide (4.7 microM); 5-aminoimidazole ribotide (4.0 microM); 4-carboxy-5-aminoimidazole ribotide (0.46 microM); N-succino-5-aminoimidazole-4-carboxamide ribotide (11 microM); 5-aminoimidazole-4-carboxamide ribotide (16 microM); 5-formamidoimidazole-4-carboxamide ribotide (2.7 microM); and IMP (57 microM). The metabolic effects of tiazofurin (25 microM) upon mouse L1210 leukemia cells growing in culture define a "metabolic crossover point" at the reaction catalyzed by IMP dehydrogenase (EC 1.1.1.205) which confirms previous reports of inhibition of this enzyme.     

Steroid 1-Dehyclrogenation by a Crude Enzyme Preparation from Arthrobacter simplex

Nonexacting purineless mutants were isolated from an inosine-forming adenine auxotroph of Bacillus pumilus. Some of them accumulated Bratton-Marshall reaction-positive material in their culture fluid. The product was isolated in crystalline form and identified with 5(4)-amino-4(5)-imidazolecarboxamide riboside (AICA-Riboside).

AICA-Riboside accumulated by the nonexacting purineless mutants was less than inosine accumulated by their parent adenine auxotroph.

A number of mutants that require adenine specifically were isolated from AICA-Riboside-forming purineless mutants. More than half of them accumulated a large amount of AICA-Riboside as compared with their parents, nonexacting purine auxotrophs. The rest of adenine-requiring mutants from purineless mutants lost the ability to accumulate AICA-Riboside.

The effect of hypoxanthine on the accumulation of ACIA-Riboside by these auxotrophs was also examined.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997     

Riboside and Ribotide of 5(or 3)-Aminopyrazole-4-carboxamide

During the investigation for dephosphorylation of 4-hydroxy-1-β-D-ribofuranosylpyrazolo-[3,4-d] pyrimidine 5′-phosphate, it was found that the compound was converted to an unknown substance by alkaline hydrolysis for 3 hr at 140°C. The structure of the substance was assigned to be 5-amino-1-β-D-ribofuranosylpyrazole-4-carboxamide 5′-phosphate. 5(or3)-Amino- pyrazole-4-carboxamide and its riboside were also obtained from 4-hydroxypyrazolo [3,4-d] pyrimidine and its riboside, respectively, under the similar conditions.

5-Amino-1-β-D-ribofuranosyipyrazole-4-carboxamide and 5-amino-1-β-D-ribofuranosyl- pyrazole-4-carboxamide 5′-phosphate are new compounds.     

Partial purine deprivation causes sporulation of Bacillus subtilis in the presence of excess ammonia, glucose and phosphate.

In strains of Bacillus subtilis able to synthesize purines de novo, massive sporulation is suppressed by the combination of excess ammonia, glucose and phosphate. Purine auxotrophs, blocked in the general or the guanine-specific portion of the branched purine pathway, sporulated in such a medium when the purine required for normal growth was removed from the medium. The resulting spore titre and the sporulation frequency increased with the residual growth rate in the purine-free medium, i.e. with the leakiness of the purine mutation. Sporulation was further increased by allowing residual growth in growth-limiting amounts of guanosine. None-leaky purine mutants blocked before 5'-phosphoribosyl-5-amino-4-imidazole carboxamide also sporulated well when supplied with 5-amino-4-imidazole carboxamide at concentrations (2 mM) that supported growth at a suboptimal rate.     

Sex-Linked Auxotrophic and Putative Auxotrophic Mutants of DROSOPHILA MELANOGASTER

Thirty-two mutants with improved growth response on a yeast-sucrose compared with a defined medium have been characterized with respect to ribonucleoside supplementability. Twenty mutants respond to either pyrimidine ribonucleoside. Four mutants respond to one or both purine ribonucleosides. Eight mutants ("putative" auxotrophs) do not respond to dietary RNA supplementation. Mapping and complementation studies suggest that eleven loci are represented: one, rudimentary, probably accounts for all pyrimidine requirers; there are three purine loci and seven at which the putative auxotrophs are found.     

Evidence that the folate-requiring enzymes of de novo purine biosynthesis are encoded by individual mRNAs

Isolation of the mRNAs encoding for the three folate-requiring enzymes involved in de novo purine biosynthesis followed by their in vitro translation resulted in three separate proteins electrophoretically identical with those previously isolated. The three enzymes are glycinamide ribonucleotide transformylase, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase, and 5,10-methenyl-, 5,10-methylene-, and 10-formyltetrahydrofolate synthetase. Thus these enzymes do not appear to be derived from large multifunctional proteins that are then subject to proteolysis in vivo or during in vitro purification. The levels of these enzymatic activities were increased by approximately 2-fold after raising the concentration of protein in the chicken's diet. The observed response is similar to that noted for glutamine phosphoribosylpyrophosphate amidotransferase, the presumed rate-limiting enzymatic activity for this pathway. For 5-amino-imidazole-4-carboxamide ribonucleotide transformylase and the trifunctional synthetase but not glycinamide ribonucleotide transformylase the increase in enzymatic activity correlates with higher mRNA levels.     

5-aminoimidazole-4-carboxamide riboside administration and purine release from the hypoxic/hypotensive rat cerebral cortex

The effects of the purine precursor 5-aminoimidazole-4-carboxamide riboside (AICAriboside) on the release of purines from cerebral cortices of normoxic and hypoxic/hypotensive rats was studied with the cortical cup technique. AICAriboside was administered either intravascularly (50 mg/kg) or intraperitoneally (500 mg/kg) to ascertain whether this agent can be used to enhance adenosine levels in the cerebral cortical interstitial fluid. Following its intraperitoneal administration AICAriboside appeared rapidly in the cortical superfusates at concentrations of up to 9 μM and remained at this level for a 90 min period. After intravascular administration, AICAriboside levels peaked at 2 μM, and then declined rapidly. No increases in basal (normoxic) or hypoxia-elicited adenosine levels in the cortical superfusates were observed. Increases did occur in the basal and hypoxia-evoked levels of hypoxanthine, xanthine and, especially, of uric acid. AICAriboside administration appears to have caused an increase in adenosine metabolite, rather than in adenosine, levels in the cerebral interstitial fluid and it may therefore be of little benefit as a precursor for adenosine formation and release in the treatment of cerebral ischemic damage.     

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.     

Structural analyses of a purine biosynthetic enzyme from Mycobacterium tuberculosis reveal a novel bound nucleotide

Enzymes of the de novo purine biosynthetic pathway have been identified as essential for the growth and survival of Mycobacterium tuberculosis and thus have potential for the development of anti-tuberculosis drugs. The final two steps of this pathway are carried out by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC), also known as PurH. This enzyme has already been the target of anti-cancer drug development. We have determined the crystal structures of the M. tuberculosis ATIC (Rv0957) both with and without the substrate 5-aminoimidazole-4-carboxamide ribonucleotide, at resolutions of 2.5 and 2.2 Å, respectively. As for other ATIC enzymes, the protein is folded into two domains, the N-terminal domain (residues 1–212) containing the cyclohydrolase active site and the C-terminal domain (residues 222–523) containing the formyltransferase active site. An adventitiously bound nucleotide was found in the cyclohydrolase active site in both structures and was identified by NMR and mass spectral analysis as a novel 5-formyl derivative of an earlier intermediate in the biosynthetic pathway 4-carboxy-5-aminoimidazole ribonucleotide. This result and other studies suggest that this novel nucleotide is a cyclohydrolase inhibitor. The dimer formed by M. tuberculosis ATIC is different from those seen for human and avian ATICs, but it has a similar ∼50-Å separation of the two active sites of the bifunctional enzyme. Evidence in M. tuberculosis ATIC for reactivity of half-the-sites in the cyclohydrolase domains can be attributed to ligand-induced movements that propagate across the dimer interface and may be a common feature of ATIC enzymes.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles. Received: 29 March 1999 / Accepted: 25 May 1999     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27?kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553?nm), in contrast to strain CE3 which produced a single 32?kDa c-type protein and did not produce the 553?nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553?nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli.