Catabolism of indole-3-acetic acid and 4- and 5-chloroindole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid. Indoleacetic acid (IAA), 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA were metabolized to different extents by strains 61A24 and 110. Metabolites were isolated and analyzed by high-performance liquid chromatography and conventional mass spectrometry (MS) methods, including MS-mass spectroscopy, UV spectroscopy, and high-performance liquid chromatography-MS. The identified products indicate a novel metabolic pathway in which IAA is metabolized via dioxindole-3-acetic acid, dioxindole, isatin, and 2-aminophenyl glyoxylic acid (isatinic acid) to anthranilic acid, which is further metabolized. Degradation of 4-Cl-IAA apparently stops at the 4-Cl-dioxindole step in contrast to 5-Cl-IAA which is metabolized to 5-Cl-anthranilic acid.

Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires addition of the substrates for induction and is oxygen dependent. The highest activity is obtained when the concentration of inducer is 0.2 mM. Spectrophotometric data are consistent with the suggestion that the indole ring is broken during degradation of IAA. We hypothesize that the enzyme catalyzes an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation product in the proposed pathway.

Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level.

The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere. Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level. The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5-6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed. Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44.

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue.

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5'-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5'-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5'-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O(2) concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O(2). 5'-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O(2) concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O(2) concentration.

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase.

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.

Enzymes of ureide synthesis in pea and soybean.

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5'-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5'-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.

Glutamine and related analogs regulate guanosine monophosphate reductase in Salmonella typhimurium.

The addition of a glutamine analog, 6-diazo-5-oxo-L-norleucine, or an inhibitor of glutamine synthetase, L-methionine-dl-sulfoximine, to the growth media of most Salmonella typhimurium strains resulted in a marked elevation of guanosine monophosphate reductase levels. The elevation caused by either compound required protein synthesis and could be antagonized by exogenous glutamine. In addition, when glutamine auxotrophs were grown in suboptimal concentrations of glutamine, the guanosine monophosphate reductase levels were increased. It is postulated that glutamine or a product of its metabolism may function under normal conditions as a negative regulatory element in the control of guanosine monophosphate reductase and that decreased effective intracellular levels of glutamine result in an increase in the level of the enzyme.