Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii

Dı́ez, Jesús; Lopez‐Ruiz, Arnaldo

doi:10.1016/0003-9861(89)90339-1

Cited by 14 publications

(9 citation statements)

References 24 publications

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“…Since none of the available Prochlorococcus strains was found to grow on nitrate, we studied the possibility of NR occurrence in cell extracts. Initial attempts were done to determine the activity of this enzyme, which can be measured with the system methyl viologen/sodium dithionite as the electron donor, using standard concentrations of all reagents as previously described [18]. However, no positive results were found.…”

Section: Resultsmentioning

confidence: 99%

“…After thawing, the samples were directly used for NR assay without further treatment, since previous studies demonstrated that no additional disruption of cells is needed to assay enzymes of Prochlorococcus [5]. NR activity was determined basically as previously described [18]. The reaction mixture contained (in order of addition): 450 μl of distilled water; 200 μl of 0.5 M phosphate buffer, pH 7.0; 100 μl of 0.2 M KNO 3 ; 100 μl of 40 mM methyl viologen; 50 μl of cell extract; and 100 μl of 0.1 M Na 2 S 2 O 4 (prepared in 0.3 M NaHCO 3 ).…”

Section: Methodsmentioning

confidence: 99%

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Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

López‐Lozano

Dı́ez

Alaoui

et al. 2002

FEMS Microbiology Ecology

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The ability to assimilate nitrate in non-axenic isolates of Prochlorococcus spp. was addressed in this work, particularly in three low-irradiance adapted strains originating from ocean depths with measurable nitrate concentrations. None of the studied strains was able to use nitrate as the sole nitrogen source. Nitrate reductase (NR; EC 1.6.6.2) activity was, however, detected using the methyl viologen/dithionite assay in crude extracts from all studied Prochlorococcus strains. Characterization of this activity unambiguously demonstrated its enzymatic origin. We observed that NR activity did not decrease in vivo under darkness. Attempts to detect the narB gene (coding for NR in other cyanobacteria) by PCR with primers designed on the basis of the specific codon usage in Prochlorococcus were unsuccessful. However, when primers were designed considering the codon frequencies typical of other bacteria, we could amplify different fragments of nas genes, coding for bacterial assimilatory NRs. Similar amplification products were obtained using colonies of contaminant bacteria from Prochlorococcus cultures as PCR template. Furthermore, NR activity was found in cultures of these contaminants, demonstrating the non-cyanobacterial origin of the enzyme. These results strongly suggest that the studied strains of Prochlorococcus lack NR, in spite of inhabiting environments with nitrate as the main nitrogen source. In addition, they indicate that the nitrite produced by heterotrophic bacteria is not transferred to Prochlorococcus for growth, thus discarding a trophic nitrogen chain between heterotrophic bacteria and Prochlorococcus in the studied cultures.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

López‐Lozano

Dı́ez

Alaoui

et al. 2002

FEMS Microbiology Ecology

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“…Isolation and Purification of the Anti-NapF Polyclonal AntibodiesPolyclonal antibodies raised against the purified NapF were obtained by following the method previously described by Diez and López-Ruiz (37). A volume of 250 l containing 135 g of purified NapF in 50 mM Tris-HCl, pH 8.0, was diluted up to 2 ml in phosphate-buffered saline buffer (135 mM NaCl, 2.6 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 8.0), mixed with 2 ml of adjuvant complete Freund (Difco Laboratories), and homogenized to obtain an uniform sample.…”

Section: Methodsmentioning

confidence: 99%

NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

Olmo-Mira

Gavira

Richardson

et al. 2004

Journal of Biological Chemistry

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The periplasmic nitrate reductase (Nap) is widespread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His 6 -tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF ؊ strain of R. sphaeroides. The His 6 -tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF ؊ strain phenotype, and the NapF-dependent reactivation of the 2,2-dipyridyltreated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.

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“…Monoraphidium brauniii N/iegeli, strain 202-7d, from the culture collection of the University of G6ttingen (Germany) was grown at 25~ in light/dark cycles (14/10 h) on 5 mM KNO3 as previously described (Diez and L6pez-Ruiz 1989). The following alternative nitrogen sources were used: potassium nitrite, ammonium sulphate, monosodium glutamate, I_-alanine and L-glutamine at a concentration of 5 mM for nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

Garcı́a-Fernández¹,

Lopez‐Ruiz

Alhama

et al. 1995

Planta

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Abstract. Glutamine-synthetase (GS; EC 6.3.1.2) activity and protein levels were measured in crude extracts from Monoraphidium braunii N~iegeli, strain 202-7d, cultures grown under different nitrogen sources. Only ammonium and L-glutamine promoted a partial enzyme inactivation, which, in the case of L-glutamine, was accompanied by a significant repression of GS. Methionine sulfoximine (MSX), a strong inhibitor of GS, produced a drastic inactivation of GS which was concomitant with a marked increase in GS protein as measured by rocket immunoelectrophoresis. Such an increase was prevented in the presence of cycloheximide. The effect of the L-glutamine analog on GS activity and protein was partially inhibited if L-glutamine was also added to cell cultures, possibly indicating competition in the transport of these two substances. In addition, the effects of MSX were reversed after longer times when cultures were treated with smaller concentrations of inhibitor. Treatment of cell cultures with azaserine, a specific inhibitor of glutamate synthase, the second enzyme acting in the ammonium assimilation pathway, promoted a strong GS inactivation and a partial repression of this enzyme, which paralleled a specific increase in the intracellular pools of glutamine High-performance liquid chromatography measurements of intracellular amino-acid concentrations showed that glutamine levels correlated negatively with GS concentration. A role for glutamine as a negative effector of GS synthesis is proposed.

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Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii

Cited by 14 publications

References 24 publications

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

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