Nitrogen assimilation defects in a mutant of Rhodopseudomonas capsulata blocked in α-ketoglutarate generation

Beatty, J

doi:10.1016/0378-1097(77)90023-4

Cited by 3 publications

(4 citation statements)

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“…Accordingly, mutants with defects in the C6 branch would be expected to require exogenous provision of KG or of L-glutamate for growth. This has been observed for mutant WE29, which lacks both CS and ICD (7). The experimental results of Fig.…”

Section: Resultssupporting

confidence: 67%

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Biosynthetic and Bioenergetic Functions of Citric Acid Cycle Reactions in Rhodopseudomonas capsulata

Beatty

Gest

1981

J Bacteriol

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Rhodopseudomonas capsulata can grow in a number of altemative modes, including (i) photosynthetic, defined here as anaerobic growth with light as the energy source, and (ii) heterotrophic, referring to aerobic heterotrophic growth in darkness. The functions of citric acid cycle sequences in these growth modes were investigated using wild-type and appropriate mutant strains. Results of growth tests and 02 utilization experiments showed that in the heterotrophic mode, energy conversion is dependent on operation of the classical citric acid cycle. Alpha-ketoglutarate dehydrogenase (KGD) activity in wild-type strain B10 is substantially higher in cells grown heterotrophically than in cells grown photosynthetically. Molecular oxygen, even at low concentration, appears to be important in regulation of KGD synthesis and, thus, in expression of citric acid cycle activity. Extracts of (photosynthetically grown) mutant strain KGD11 lack demonstrable KGD activity, and in contrast to the wild type, KGD11 is unable to grow heterotrophically on succinate, malate, or pyruvate owing to failure of the energy conversion function of the citric acid cycle. KGD11, however, grows well photosynthetically on malate or on CO2 + H2. The KGD activity level required to support the bioenergetic function of the citric acid cycle is evidently much higher than that necessary to satisfy biosynthetic demands; thus, a very low rate of succinyl-coenzyme A formation (needed for biosynthesis) in the mutant would suffice for growth under photosynthetic conditions. In wild-type R. capsulata, the a-ketoglutarate required for glutamate synthesis is ordinarily generated via citric acid cycle reactions, which include the conversions catalyzed by citrate synthase and isocitrate dehydrogenase. Mutants blocked in the former or both of these enzymes can grow photosynthetically if provided with an exogenous source of a-ketoglutarate or glutamate, but grow very poorly (if at all) as heterotrophs since the energy supply under these conditions depends on operation of the complete citric acid cycle.

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Section: Resultssupporting

confidence: 67%

“…The general characteristics of R. capsulata wild-type strain B10 are typical for the species (36). Mutant WE29 is a derivative of B10, and lacks both citrate synthase (CS) and isocitrate dehydrogenase (ICD) activities (7). Although this strain was originally thought to be a single-site mutant, more detailed genetic analysis (J. T. Beatty, M.A.…”

Section: Methodsmentioning

confidence: 99%

Biosynthetic and Bioenergetic Functions of Citric Acid Cycle Reactions in Rhodopseudomonas capsulata

Beatty

Gest

1981

J Bacteriol

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“…A mutant of Rb. capsulatus has been isolated which is unable to grow in the absence of glutamate or a-ketoglutarate; this mutant was found to lack both citrate synthase and isocitrate dehydrogenase activity [219]. Spontaneous revertants which could grow in the absence of a source of a-ketoglutarate could not be isolated, but the wild-type phenotype could be restored by treatment with gene transfer agent.…”

Section: Nh4 + Assimilationmentioning

confidence: 99%

Biochemical genetics revisited: the use of mutants to study carbon and nitrogen metabolism in the photosynthetic bacteria

Willison¹

1993

FEMS Microbiology Letters

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The biochemical genetics approach is defined as the use of mutants, in comparative studies with the wild‐type, to obtain information about biochemical and physiological processes in complex metabolic systems. This approach has been used extensively, for example in studies on the bioenergetics of the photosynthetic bacteria, but has been applied less frequently to studies of intermediary carbon and nitrogen metabolism in phototrophic organisms. Several important processes in photosynthetic bacteria—the regulation of nitrogenase synthesis and activity, the control of intracellular redox balance during photoheterotrophic growth, and chemotaxis—have been shown to involve metabolism. However, current understanding of carbon and nitrogen metabolism in these organisms is insufficient to allow a complete understanding of these phenomena. The purpose of the present review is to give an overview of carbon and nitrogen metabolism in the photosynthetic bacteria, with particular emphasis on work carried out with mutants, and to indicate areas in which the biochemical genetics approach could be applied successfully. In particular, it will be argued that, in the case of Rhodobacter capsulatus and Rb. sphaeroides, two species which are fast‐growing, possess a versatile metabolism, and have been extensively studied genetically, it should be possible to obtain a complete, integrated description of carbon and nitrogen metabolism, and to undertake a qualitative and quantitative analysis of the flow of carbon and reducing equivalents during photoheterotrophic growth. This would require a systematic biochemical genetic study employing techniques such as HPLC, NMR, and mass spectrometry, which are briefly discussed. The review is concerned mainly with Rb. capsulatus and Rb. sphaeroides, since most studies with mutants have been carried out with these organisms. However, where possible, a comparison is made with other species of purple non‐sulphur bacteria and with purple and green sulphur bacteria, and recent literature relevant to these organisms has been cited.

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“…The mutation in Fos252, which leads to a requirement for a-ketoglutarate or glutamate for good growth, could affect either citrate synthase as in the gitA (citrate synthase) mutants of E. coli (1,11) or isocitrate dehydrogenase (15) or both enzymes (3,15). Complementation of Fos252 by the gitA gene from E. coli conclusively demonstrates the first alternative to be correct, as was first indicated by the low activities of this enzyme in the mutant.…”

Section: Discussionmentioning

confidence: 99%

Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum

Ramos¹,

Robson²

1985

J Bacteriol

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A class of Azotobacter chroococcum mutants induced by Tn1 that were defective in normal aerobic nitrogen fixation when grown on sugars (Fos-) were corrected by provision of alpha-ketoglutarate or glutamate. In a representative mutant, Fos252, rates of evolution of 14CO2 from [14C]acetate or [14C]glucose were 5% of the parental values, although uptake and incorporation were normal for both substrates. The results suggest that a lesion affects the entry of substrates into the tricarboxylic acid cycle. The activity of citrate synthase in Fos252 in vitro was 5% that of the parents. The citrate synthase (gltA) gene from Escherichia coli was cloned into broad-host-range vectors and mobilized into Fos252. The plasmids restored parental citrate synthase activities to Fos252 and complemented the inability to fix N2 in air. The data indicate that a mutation causing an intrinsic limitation in respiratory capacity abolishes normal aerobic N2 fixation, which is consistent with the hypothesis of respiratory protection for nitrogenase in Azotobacter species.

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Nitrogen assimilation defects in a mutant of Rhodopseudomonas capsulata blocked in α-ketoglutarate generation

Cited by 3 publications

References 1 publication

Biosynthetic and Bioenergetic Functions of Citric Acid Cycle Reactions in Rhodopseudomonas capsulata

Biosynthetic and Bioenergetic Functions of Citric Acid Cycle Reactions in Rhodopseudomonas capsulata

Biochemical genetics revisited: the use of mutants to study carbon and nitrogen metabolism in the photosynthetic bacteria

Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum

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