Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN ofRhizobium meliloti involved in symbiotic nitrogen fixation

Masepohl, Bernd; Kutsche, Michael; Riedel, Kai‐Uwe; Schmehl, Manfred; Klipp, Werner; Pühler, Alfred

doi:10.1007/bf00587558

Cited by 23 publications

(17 citation statements)

References 33 publications

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“…This may explain why we have not found extra residues with less than 6 residues among the natural ferredoxins of the subclass. Our results confirm the report by Masepohl et al (21), who showed that the deletion and substitution of the extra residues of R. meliloti FdxN has almost no effect on the capacity to support nitrogen fixation in planta. On the other hand, Rhodospirillum rubrum is reported to produce FdxN protein even under nif-repressed growth conditions (11).…”

Section: Discussionsupporting

confidence: 93%

“…A gene to encode a ferredoxin with similar extra residues has also been identified in the non-diazotrophic bacterium H. influenzae (20). These findings, together with the results of the protein engineering studies with R. meliloti (21) and R. capsulatus (this study), suggest that the possession of loop-out and extra residues in the second clus- ter-binding motif does not necessarily indicate a functional link to nitrogen fixation.…”

Section: Discussionsupporting

confidence: 56%

“…At the physiological level, the site-specific mutagenesis of R. meliloti FdxN has shown that the substitution of the additional Cys to Ser abolishes the capacity to support nitrogen fixation in planta, while the deletion of the extra residues has little effect (21). At the structural level, the nuclear magnetic resonance (NMR) analyses of the Chromatium vinosum ferredoxin, which has a unique COOH-terminal extension of 22 amino acids, have revealed that this extension sequence forms an ␣-helix and interacts with a turn formed by the extra residues between the Cys residues.…”

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confidence: 99%

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Site-specific Mutagenesis of Rhodobacter capsulatus Ferredoxin I, FdxN, That Functions in Nitrogen Fixation

Saeki

Tokuda

Fukuyama

et al. 1996

Journal of Biological Chemistry

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One of the two [4Fe-4S]-type clusters of the 41 and Cys 50 also yielded active products. Second, three of the modified FdxN proteins were subjected to purification. Only the GA protein, whose 8 residues between positions 42 and 49 were replaced by the GlyAla sequence, was purified. The GA protein and the authentic FdxN showed similar optical properties. The two clusters in the former had E m values of ؊490 and ؊430 mV, while those in the latter had an identical value of ؊490 mV, when determined by EPR analysis. It was concluded that: 1) Cys 59 is not a ligand to [4Fe-4S] clusters but is important for structural integrity, 2) the residues between positions 42 and 49 may form a "loop-out" from a structure analogous to the Peptococcus aerogenes ferredoxin, and 3) the loop-out region does not have functional significance in nitrogen fixation but may be responsible for maintaining the highly negative redox potential of one of the two clusters.Ferredoxins are generally small iron-sulfur proteins that function in diverse metabolic pathways. They can be divided into several classes depending on the nature and number of the cluster is chelated by the first three Cys residues of one motif and the last Cys residue of the other motif. These properties reflect on their tertiary structures that display an approximate internal 2-fold rotation axis to relate the two clusters and most of the polypeptide chain (3-5).Among the 2[4Fe-4S]-type ferredoxins, there is a subclass in that the second cluster-binding motif conforms to a distinct sequence consensus, Cys-X 2 -Cys-X 7ϳ9 -Cys-X 3 -Cys-X 3ϳ5 -Cys, as depicted in Fig. 1 (1). This consensus can be characterized by two features: 1) possession of "extra" residues, where the number of residues between second and third Cys is larger than the common number two; and 2) possession of an "additional" fifth Cys, which can be a potential ligand to the Fe-S cluster. Members of this subclass have been purified from photosynthetic bacteria that are diazotrophs (6 -11). In addition, several members have also been identified as products of potential ferredoxin genes, which have often been designated as fdxNs, in the nif (or anf) gene region of photosynthetic (12) and non-photosynthetic diazotrophs (13-17). Here, we tentatively designate this subclass as photosynthetic bacterial and nif-associated ferredoxins. The distribution among such bacterial species and the results of gene disruption experiments performed with Bradyrhizobium japonicum (13), Rhizobium meliloti (15), and Rhodobacter capsulatus (18, 19) may lead to the inference that these members have a function(s) in nitrogen fixation. Nevertheless, a gene to encode a ferredoxin of this subclass is also identified in a non-diazotrophic bacterium Haemophilus influenzae, whose whole nucleotide sequence of the genome has recently been determined (20).It is of interest if the above two characteristics of the unique second cluster-binding motif have physiological and/or physicochemical significance, but there have been only a few studies on this...

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 56%

mentioning

confidence: 99%

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Site-specific Mutagenesis of Rhodobacter capsulatus Ferredoxin I, FdxN, That Functions in Nitrogen Fixation

Saeki

Tokuda

Fukuyama

et al. 1996

Journal of Biological Chemistry

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“…The Escherichia coli strains KAM3 (kindly provided by T. Tsuchiya, Okayama, Japan) containing a deletion in the multidrug efflux gene region acrAB (22), DH5␣ mcr (11), S17-1 (31), and XL1-Blue (5) were grown at 37°C in Luria broth (LB) medium. Sinorhizobium meliloti 2011 (fdxN::Tc) was grown in TY medium (20). Selection for plasmids that mediate fluoroquinolone-resistance was done on agar plates containing 0.04 g of norfloxacin per ml.…”

Section: Methodsmentioning

confidence: 99%

“…The pGNB2 derivative pGNB2-aphII was transferred into the mobilisator strain E. coli S17-1 containing an integrated derivative of the IncP-1␣ plasmid RP4, providing transfer functions in trans by transformation (31). E. coli S17-1(pGNB2-aphII) was mated with the tetracycline-resistant E. coli strain XL1-Blue (5) and the tetracycline-resistant ␣-proteobacterium Sinorhizobium meliloti 2011 (fdxN::Tc) (20) under conditions described previously (33). Plasmid pGNB2-aphII-containing transconjugants were selected on media containing, respectively, 5 g of tetracycline ml Ϫ1 and 120 g of neomycin ml Ϫ1 for S. meliloti 2011 and 5 g of tetracycline ml…”

Section: Methodsmentioning

confidence: 99%

Mobilizable IncQ-Related Plasmid Carrying a New Quinolone Resistance Gene, qnrS2 , Isolated from the Bacterial Community of a Wastewater Treatment Plant

Bönemann

Stiens

Pühler

et al. 2006

Antimicrob Agents Chemother

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Plasmid-encoded quinolone resistance was previously reported for different bacteria isolated from patients not only in the United States and Asia but also in Europe. Here we describe the isolation, by applying a new selection strategy, of the quinolone resistance plasmid pGNB2 from an activated sludge bacterial community of a wastewater treatment plant in Germany. The hypersensitive Escherichia coli strain KAM3 carrying a mutation in the multidrug efflux system genes acrAB was transformed with total plasmid DNA preparations isolated from activated sludge bacteria and subsequently selected on medium containing the fluoroquinolone norfloxacin. This approach resulted in the isolation of plasmid pGNB2 conferring decreased susceptibility to nalidixic acid and to different fluoroquinolones. Analysis of the pGNB2 nucleotide sequence revealed that it is 8,469 bp in size and has a G؉C content of 58.2%. The plasmid backbone is composed of a replication initiation module (repA-repC) belonging to the IncQ-family and a two-component mobilization module that confers horizontal mobility to the plasmid. In addition, plasmid pGNB2 carries an accessory module consisting of a transposon Tn1721 remnant and the quinolone resistance gene, qnrS2, that is 92% identical to the qnrS gene located on plasmid pAH0376 from Shigella flexneri 2b. QnrS2 belongs to the pentapeptide repeat protein family and is predicted to protect DNA-gyrase activity against quinolones. This is not only the first report on a completely sequenced plasmid mediating quinolone resistance isolated from an environmental sample but also on the first qnrS-like gene detected in Europe.Quinolones and especially fluoroquinolones are among the most often prescribed antimicrobial drugs worldwide (1). As a consequence, high resistance rates have developed due to the persisting selection pressure. Resistances are mainly attributed to chromosomal mutations in the genes gyrA and parC, which encode, respectively, subunit A of DNA-gyrase (GyrA) and topoisomerase IV (ParC), representing the target enzymes for quinolones (25). Nevertheless, plasmid-encoded quinolone resistance is of great concern since these resistance determinants potentially can be disseminated among bacteria due to plasmid mobility. The isolation of the multiresistance plasmid pMG252 from a clinical strain of Klebsiella pneumoniae in Birmingham, Alabama, in 1994 was the first documented discovery of a plasmid-encoded resistance to quinolones (19). The quinolone resistance gene qnrA, located on pMG252, encodes a 218-amino-acid protein of the pentapeptide repeat family. Members of this protein family are characterized by the repetition of the consensus sequence (C/A)-(D/N)-(L/F)-X-X (3). It was shown that Qnr protects DNA-gyrase and topoisomerase IV activity against inhibition by quinolones (38-40). Recently, other qnr genes, namely, qnrS and qnrB, were identified on plasmids originating from clinical isolates (12, 16).Several multiresistance plasmids were previously isolated from the activated sludge bacterial com...

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Electron Transport to Nitrogenase: Diverse Routes for a Common Destination

Saeki

Nitrogen Fixation: Origins, Applications, and Research Progress

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Nitrogenase, which consists of dinitrogenase (e.g., the MoFe protein) and dinitrogenase reductase (the Fe protein), catalyzes the biological reduction of molecular nitrogen (N 2 ) according to the following equation: N 2 + (6+2n) H + + (6+2n) e + (12+4n) MgATP 2 NH 3 + n H 2 + (12+4n) MgADP + (12+4n) Pi (where n 1 depending on the availability of both H + and e )Electrons are first transferred to dinitrogenase reductase, which in turn donates electrons to dinitrogenase. This reaction is common to all three conventional nitrogenases; the molybdenum-containing nitrogenase as well as the alternative vanadium-containing and the iron-only nitrogenases. It does not apply to the unusual N 2 -fixation system reported for the thermophile, Streptomyces thermoautotrophicus, which was isolated from a burning charcoal pile (Gadkari et al., 1992;Ribbe et al., 1997). As shown in the above equation, the availability of reducing power is as important as the availability of ATP for the throughput of the nitrogenase reaction. When the supply of reducing power is limited, the value of n increases (Haaker and Klugkist, 1987) and, as a consequence, net fixation of N 2 decreases, with more reducing equivalents diverted towards H 2 production.

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Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN ofRhizobium meliloti involved in symbiotic nitrogen fixation

Cited by 23 publications

References 33 publications

Site-specific Mutagenesis of Rhodobacter capsulatus Ferredoxin I, FdxN, That Functions in Nitrogen Fixation

Site-specific Mutagenesis of Rhodobacter capsulatus Ferredoxin I, FdxN, That Functions in Nitrogen Fixation

Mobilizable IncQ-Related Plasmid Carrying a New Quinolone Resistance Gene, qnrS2 , Isolated from the Bacterial Community of a Wastewater Treatment Plant

Electron Transport to Nitrogenase: Diverse Routes for a Common Destination

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