Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Stam, H.; Verseveld, Henk W. van; Vries, Wytske de; Stouthamer, A. H.

doi:10.1007/bf00692712

Cited by 49 publications

(41 citation statements)

References 38 publications

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“…As reported here, the Azorhizobium coxA gene was most highly expressed when cells grew rapidly in 02-rich, nutrient-rich environments. Indeed, in Azorhizobium chemostat cultures, cytaa3 activity also decreased as dissolved 02 tension decreased (40). Consistent with the regulation of coxA gene expression, little cytaa3 activity was evident in spectra from late exponential-phase batch cultures.…”

Section: Discussionmentioning

confidence: 67%

“…For oxidative phosphorylation, cytc-dependent respiration is most efficient and, a priori, best suited for the high energy demands of N2 fixation (1,31,40). Unlike those of quinol oxidases, cytc respiratory chains employ an additional, cytbcl complex which augments proton pumping.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, cyto signals are present in the spectra of bacteroids taken from several Rhizobium spp. (3,40). Because pleiotropicAzorhizobium cytc mutants used both cyto and cytd as terminal oxidases and grew normally in defined media, cyto appears to be a quinol oxidase, as is true for enteric bacterial cyto.…”

Section: Discussionmentioning

confidence: 99%

“…For these two disparate N2 fixation processes, dissolved 02 optima vary some 3 orders of magnitude: 10 ,uM in culture versus 10 nM in planta (5, 9). By spectrophotometry, Azorhizobium continuous cultures actively fixing N2 show cyto, cytd, and cytaa3 activities (40). To analyze their physiological roles, we here report the construction of Azorhizobium cytochrome oxidases by reverse genetics.…”

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confidence: 99%

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Azorhizobium caulinodans respires with at least four terminal oxidases

Kitts

Ludwig

1994

J Bacteriol

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In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to 02. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory edonors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 401% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70%6 lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e -donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allowAzorhizobium spp. to flourish in wide-ranging 02 environments.As biological N2 fixation is extremely 02 sensitive, N2-fixing organisms have evolved 02 detoxification and/or avoidance mechanisms. Azotobacter spp., free-living aerobes, elaborate cells with very high total membrane surface area and, consequently, very high concentrations of respiratory electron (e-) transport complexes. For respiration, Azotobacter spp. employ cytochrome d (cytd) and cytochrome o (cyto) quinol oxidases but not cytc oxidases (20,44). To fix N2 in air, Azotobacter spp. require cytd oxidase (22), which might also mitigate oxidative ...

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Azorhizobium caulinodans respires with at least four terminal oxidases

Kitts

Ludwig

1994

J Bacteriol

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“…A nitrosoguanidine-induced mutant with increased TMPD oxidase activity when cultured under microaerophilic conditions (TMPD++) expressed cytochrome aa3 constitutively and displayed premature symbiotic nitrogen fixation activity (18). In other Rhizobium species, cytochrome aa3 is not expressed when the cells are cultured at low 02 tensions (4,20) or during symbiosis (1).…”

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confidence: 99%

Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3

et al. 1990

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Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3). Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells. Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3.

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Construction of an R‐prime plasmid carrying the hup genes of Azorhizobium caulinodans ORS571 and its transfer to and stability in Rhizobium meliloti

Miksch

Lentzsch

1990

J. Basic Microbiol.

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An R-prime plasmid (pJB3JI-18) carrying a 10 kb region of ,4iorhiiohiun1 c~uu/inodurrs ORSS71 DNA which codes for uptake hydrogenase activity was isolated from a Km' derivative of R68.45. R-prime plasmids were obtained by transferring R68.45 to ORSS71. retransferring it to E. coli. and selecting transconjugants for hydrogenase activity by means of a colour test based on methylene bluc. The R-prime plasmid DNA was hybridized with a DNA region which contained the Imp genes of Brarlrrliizohium juponicuiii, indicating homology between the heterologous lzirp genes. Transfer of the R-prime plasmid to different Hup-mutants restored the Hup' phenotype. When it was introduced t o R. meli/ori strains, it maintained its molecular weight and did not affect indigenous plasmids in two of three recipients. In the third recipient cointegrate formation and incompatibility with indigenous plasmids were found. Molecular hydrogen is a by-product in the biological reduction of dinitrogen to ammonia. The formation of hydrogen by nitrogenase implies loss of energy and reduction equivalents. In bacteroids, hydrogen formation may consume 40 to 60"; of the electron flux to nitrogenase (SCHUBERT and EVANS 1976). A number of nitrogen-fixing Rltizobiutn strains reoxidize hydrogen via an hydrogen uptake system. In most cases the hydrogenase activity is insufficient to recycle all the hydrogen formed. Previously, the fast-growing strain ORS571 of Azorhizobiunz caulinodans was isolated from stem nodules of the tropical legume Sesbania rostrata (DREYFUS and DOMMERGUES 1981). This strain forms nodules on both the stem and the roots of the host plant. Nitrogen-fixing cultures of ORS571 show considerable hydrogenase activity (STAM et al. 1984). In this special case, hydrogen oxidation is coupled with ATP formation, which is not found in other Rhizobiunz species or strains (NELSON 1983). These features prompted us to look for suitable cloning procedures, because the transfer of the /?up genes of ORS571 to other rhizobia, e.g. R. meliloti would be beneficial for improving nitrogen fixation. Transmissible plasmids carrying DNA segments have been isolated in several bacterial species by using in vivo procedures. These prime plasmids have been of great value in the study of chromosomal genes (Low 1972, HOLLOWAY 1979 HOLLOWAY 1976) to induce recombination with the chromosome was expoited in these studies. ORS571 is a plasmidless strain, and therefore the /zup genes must be located on the chromosome.Here we report the isolation and characterization of an R-prime plasmid containing a 10 kb fragment of A . caufinodans ORS571 DNA. This fragment includes the hup genes.Moreover, we show the stability of the R-prime plasmid after transfer to several R. tneliloti strains and the expression of the hup genes in this species.

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Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Cited by 49 publications

References 38 publications

Azorhizobium caulinodans respires with at least four terminal oxidases

Azorhizobium caulinodans respires with at least four terminal oxidases

Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3

Construction of an R‐prime plasmid carrying the hup genes of Azorhizobium caulinodans ORS571 and its transfer to and stability in Rhizobium meliloti

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