Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J. M., and Burgess, B. K.(1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon.

vealed that there was another small acidic protein that was dramatically overproduced in the FdI Ϫ strain when compared to the wild-type. This protein was given the trivial name Protein X (11,12). The observation that the synthesis of Protein X was "repressed" by FdI further led to the proposal that FdI might be a novel DNA binding repressor protein (13). Thus, FdI appears to have both an electron transfer and a regulatory function. In order to gain insight into both of these functions, we previously reported the purification and characterization of Protein X (14). It was shown to be a M r ϳ 29,000, NADPHspecific flavoprotein whose physical properties and NH 2 -terminal amino acid sequence showed striking similarity with the NADPH:ferredoxin reductase from Escherichia coli (14). Protein X was further shown to bind FdI specifically, suggesting that the two proteins were likely to be redox partners in vivo (14).
Here we report the cloning and sequencing of the A. vinelandii gene encoding Protein X and the overexpression of the protein product.

EXPERIMENTAL PROCEDURES
Materials-AvFdI was purified and crystallized (2,8) and A. vinelandii NADPH:ferredoxin reductase was purified (14) as described previously. Polyclonal antibodies against both proteins were raised at Bethyl Laboratories, Montgomery, TX. The restriction enzymes MboI and SfuI, T 4 DNA ligase, T 4 polynucleotide kinase, proteinase K, 5-bromo-4-chloro-3-indolyl-␤-D-galactoside, and isopropyl-1-thio-␤-D-galactoside were from Boehringer Mannheim. Tris, Sephadex G-25, DNase, RNase, ampicillin, kanamycin, streptomycin, N-Z-Amine A, and XAR Kodak film were from Sigma. All other restriction endonucleases, the silver sequencing kit, the Erase-a-Base kit, and the Wizard Miniprep kit were from Promega. The vector EMBL-3 and the Gigapack Gold packaging kit were from Stratagene. [␥-32 P]ATP (3000 Ci/mmol) was obtained from Amersham or DuPont NEN. The universal and reverse sequence primers were obtained from the Auto-Read TM sequencing kit from Pharmacia. Nitrocellulose filters (S&S NC TM ) were purchased from Intermountain Scientific, and "Hybond-N" nylon for Southern analysis was from Amersham. All oligonucleotides were purchased from the Midland Reagent Corp. Bacterial strain DH5␣FЈ was a gift from Prof. M. O'Connor, and the plasmids pBluescript II SK(Ϫ) and pBlue-scriptII KS(ϩ) were generous gifts from Prof. E. Wagner, both from the Dept. of Molecular Biology and Biochemistry at the University of California, Irvine.
Construction and Screening of an A. vinelandii Genomic Library-A. vinelandii DNA was partially digested with MboI, 15-kb 1 fragments were isolated from sucrose gradients (15), and a library was constructed in the vector EMBL-3 and packaged with a Gigapack Gold packaging kit. Ten plates (137 mm in diameter) of NZY media (15), with ϳ1000 plaques/plate on E. coli strain P2392 were prepared. After 12 h of growth at 37°C, replicate lifts from each plate were prepared using nitrocellulose filters. These filters were hybridized using the ␥-32 P-endlabeled probe, A. vinelandii fpr oligonucleotide 1 (see "Results and Discussion") and were subjected to two 15-min washes with 0.2 ϫ SSC and O.1% SDS at 25°C (15). (The oligonucleotides were end-labeled with [␥-32 P]ATP by T4 polynucleotide kinase.) Excess label was removed using Sephadex G-50 spin columns (15), and the filters were * This work was supported in part by National Institutes of Health Grant RO1-GM45209 (to B. K. B.) with a Minority Supplement (to J. M. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) L36319.
‡ Supported in part by a biotechnology metalloprotein training grant from the University of California.
§ To whom correspondence should be addressed. Tel.: 714-824-4297; Fax: 714-824-8551. exposed on Kodak XAR film overnight. From the primary screening, 10 plaques were selected and treated to a second round of screening. The secondary screening resulted in 10 pure plaques which were stored in 1 ml of SM buffer (15) and 20 l of chloroform.
Construction of Subclonesphage DNA containing the A. vinelandii fpr gene was purified from the 10 positive plaques using published methods (16). The DNA was digested with SalI, subjected to agarose gel electrophoresis, transferred onto nitrocellulose filters, and hybridized to oligonucleotide 1. A 1.5-kb SalI fragment containing the fpr gene was identified and subsequently gel-purified from agarose gels using the Geneclean method. This SalI fragment was then subcloned into SalIdigested pBluescriptII SK(Ϫ) using T4 DNA ligase and transformed into competent DH5␣FЈ E. coli cells (15). The transformants were screened for ampicillin resistance and blue-white selection. Recombinant DNA was purified using the Wizard Miniprep kit.
DNA Sequence Determination and Southern Blot Analysis-The A. vinelandii fpr gene was sequenced in both directions from the 1.5-kb insert that was subcloned into two versions of pBluescript II, SK(Ϫ) (pS-XF1.5Sal) and KS(ϩ) (pK-XF1.5Sal). Both sequences were in agreement. Nested deletions were constructed in both vectors using the Erase-a-Base kit. Each of the clones was sequenced twice with universal or reverse primers from the Pharmacia Auto-Read sequencing kit. The resulting reaction products were resolved and processed on an Automated Laser Fluorescence (Pharmacia) sequencer. For Southern analysis, A. vinelandii strain OP chromosomal DNA was isolated, digested to completion with restriction endonucleases, subjected to agarose gel electrophoresis, transferred to nylon filters, and hybridized (15) to a 1.2-kb XhoI 32 P-labeled fragment containing the entire fpr coding region.
Construction of a NADPH:Ferredoxin Reductase Overexpression Strain-The fpr gene was subcloned into a broad host-range multicopy plasmid pKT230 which was used previously to successfully overexpress FdI (17). For this study, pS-XF1.5Sal (Fig. 1) and pKT230 (17) were both digested with XhoI and then the two fragments were ligated together and transformed into E. coli strain DH5␣FЈ (15) which was screened for kanamycin sensitivity and streptomycin resistance. The subcloned DNA was then isolated by using Promega's Wizard Minipreps kit, and the orientation of the A. vinelandii DNA insert was determined by restriction analysis. The pKT230 derivatives with the gene in the two orientations were designated pKTF1.2 and pKTR1.2. These plasmids were then introduced into the A. vinelandii wild-type trans-strain using electroporation as described by BTX Inc. Following electroporation, the A. vinelandii cells were recovered for 5 h in 1 ml of Burk's media supplemented with 36 mM ammonium acetate at 30°C with shaking at 200 rpm. The cells were then plated on selective Burk's media minus ammonium acetate supplemented with 0.1 g/ml streptomycin.

RESULTS AND DISCUSSION
Cloning and Subcloning of the A. vinelandii NADPH:Ferredoxin Reductase Gene-We recently published the NH 2 -terminal amino acid sequence of a NADP ϩ /NADPH-specific flavoprotein that is overexpressed in FdI Ϫ strains of A. vinelandii (14). We selected a region of this amino acid sequence for the synthesis of the corresponding oligonucleotide probe. The probe (5Ј-CACCACTGGAACGA(CT)ACCCT(GC)TTCTCCTTCAAG-ACCACCCG(CT)AACCC-3Ј), a 47-mer (degenerate in 3 places), was based on residues 13-28 of the protein sequence (14) and the known codon bias of this G-C-rich organism (18). This oligonucleotide, end-labeled with ␥-32 P, was used to screen a library of SalI-digested A. vinelandii DNA. Following initial screening of 10,000 plaques, 10 positive plaques were selected. Purified DNA was digested with SalI, and a 1.5-kb fragment which hybridized to the oligonucleotide was selected. This fragment was then subcloned into Bluescript as described under "Experimental Procedures." Fig. 1 is a restriction map of the A. vinelandii DNA portion of the resulting plasmid designated pS-XF1.5Sal.
DNA Sequence Analysis-The complete nucleotide sequence of the A. vinelandii DNA fragment contained in pS-XF1.5Sal ( Fig. 1) was determined by making unidirectional deletions of pS-XF1.5Sal and pKϩXF1.5Sal and subsequently sequencing selected deletion clones. The sequencing strategy is shown in  shown in Fig. 3. As discussed in detail below, the predicted protein sequence is similar to that of the E. coli fpr gene product (19). On the basis of this amino acid sequence similarity and the previously reported physical similarities (14), the gene shown in Fig. 3 has been designated the fpr gene of A. vinelandii. The amino acid sequence derived from the DNA sequence (Fig. 3) is in perfect agreement with the previously determined NH 2 -terminal 35-amino-acid residue protein sequence (14) demonstrating that the gene encoding the protein of interest has been cloned.
Homology of the A. vinelandii fpr Gene Product with the E. coli fpr Gene Product- Table I gives the results of an exhaustive search of the GenBank, EMBL, PIR-Protein, and Swiss-PROT data bases using the sequence shown in Fig. 3. The data show that the A. vinelandii protein is closely related to a family of ferredoxin oxidoreductases that fall into two general classes. The plant ferredoxin NADP ϩ reductases, which catalyze the last step in photosynthesis to yield NADPH, are designated FNR and have an NH 2 -terminal extension that serves as a targeting sequence for localizing these proteins to the chloroplasts. The bacterial NADPH:ferredoxin reductases are missing the NH 2 -terminal extension and are designated FPR because the FNR designation had already been used for an unrelated protein in E. coli (19). As shown in Table I, the A.
vinelandii protein falls into the bacterial class. Fig. 4 compares the sequence of the A. vinelandii fpr gene product reported here to that of the previously published E. coli fpr gene product (19). When these two sequences are optimally aligned, 33% of the amino acids are identical and the overall similarity is 53%. In E. coli, the fpr gene product was originally designated dA1 and was proposed to be a NADPH:ferredoxin reductase based on its similarity to the spinach enzyme (19). We have previously compared the physical properties of the protein products from E. coli and A. vinelandii and have shown that both proteins have molecular weights of ϳ29,000, that both contain noncovalently bound FAD, that both are specific for NADPH, and that both catalyze the ferredoxin-dependent reduction of cytochrome c (14). On this basis, and with the additional sequence information shown in Fig. 4, we now designate the A. vinelandii fpr product (which was formerly given the trivial name Protein X (11)) as A. vinelandii NADPH: ferredoxin reductase (ferredoxin-NADP ϩ reductase, EC 1.18.1.2).
As shown in Fig. 4, the similarity of the A. vinelandii protein to the E. coli NADPH:ferredoxin reductase occurs over the entire length of the proteins to yield products of similar molecular weight. A recent structural study of an extended family of flavoprotein reductases further identified six sequence motifs that are highly conserved among all members of the family and are involved in FAD and NADPH binding (20). As shown in Fig.  5, all six of these regions are also highly conserved when the E. coli and A. vinelandii sequences are compared, confirming that the proteins are likely to be closely related functionally.
A. vinelandii Has Only a Single Copy of the fpr Gene-  Because A. vinelandii has multiple copies of some genes (21), we examined whether the fpr gene was also reiterated on the wild-type A. vinelandii chromosome. To accomplish this, Southern analysis was performed using a 1.2-kilobase XhoI restriction enzyme fragment containing the fpr gene as a probe of A. vinelandii genomic DNA digested with various restriction enzymes. As shown in Fig. 6, in each case where the enzyme used did not cut within the gene, the fpr probe hybridized to only a single restriction enzyme fragment in the respective Southern analysis. These results show that there is only a single fpr encoding sequence on the A. vinelandii chromosome.
Relationship to N 2 Fixation-When A. vinelandii is grown under N 2 -fixing conditions, the enzyme nitrogenase receives electrons directly from reduced flavodoxin (Fld), however, the mechanism of Fld reduction in this organism has not been established (22,23). Although E. coli does not fix nitrogen, it does contain a Fld which is used as an electron donor in anaerobic metabolism (24 -26). In that organism, the fpr gene product (NADPH:ferredoxin reductase) appears to serve as the immediate electron donor to Fld (19,26), suggesting that in A. vinelandii the fpr gene product might similarly serve to mediate electron transfer between NADPH and Fld. In A. vinelandii, however, the reduced Fld could then be used to support N 2 reduction by nitrogenase. In support of that idea, we observed during the purification of A. vinelandii NADPH:ferredoxin reductase (14) and by Western blot analysis (Fig. 7) that the protein was up-regulated when the cells were grown under N 2 -fixing conditions. It is interesting to note that Klugkist et al. (27) previously reported that in A. vinelandii a NADPH-specific 29,000-dalton membrane-bound electron transport protein was up-regulated under N 2 -fixing conditions. It is possible that their membrane-bound protein and the soluble protein reported here are the same, and that the reported differences in solubility arise from the different methods used to rupture the cells.
In spite of obvious up-regulation of the NADPH:ferredoxin reductase under N 2 -fixing conditions (Fig. 7), the restriction fragments shown in Fig. 1, and the sequence shown in Fig. 3, which includes the fpr gene that codes for A. vinelandii NAD-PH:ferredoxin reductase, are not found in the 50 kb of A. vinelandii DNA from the nif region that has been mapped to date. All nif genes so far sequenced from Azotobacter and Klebsiella contain a consensus nif promotor sequence and a nifA binding site (e.g. Ref. 21). This nif promotor sequence is not found immediately upstream of the fpr gene nor is a nifA binding site found within 400 bp upstream of the fpr initiation codon (Fig. 3). This situation is reminiscent of the situation with respect to the regulation of cytochrome d in A. vinelandii  (28). Cytochrome d is involved in maintaining the low intracellular O 2 concentrations that are required for N 2 fixation. The gene encoding cytochrome d is not located in the nif region of the chromosome, and it is not preceded by a nif specific promoter or nifA binding sequence. However, like the fpr gene (Fig. 7), the cytochrome d gene is up-regulated when the cells are grown under N 2 -fixing conditions (28).
Further analysis of the regions upstream of the fpr gene shows that two putative 70 type E. coli consensus promotor elements are present (Fig. 3). The data in Fig. 3 also show an excellent ribosome binding site (29) for A. vinelandii NADPH: ferredoxin reductase 12 bases upstream from the initiation codon. Downstream of the coding region there is a predicted stem-loop structure denoted by the arrows (Fig. 3) which probably acts as a rho-independent transcription termination signal (30). Thus, it is unlikely that additional genes are in the same operon downstream of fpr.
Overexpression of A. vinelandii NADPH:Ferredoxin-Reductase in A. vinelandii-Wild-type cells of A. vinelandii synthesize only very small quantities of NADPH:ferredoxin reductase. To proceed with our crystallography studies (31) and other experiments, larger quantities of purified protein were required. In order to overproduce the NADPH:ferredoxin reductase in its native background, multiple copies of the fpr gene were maintained in a wild-type strain of A. vinelandii (designated trans) using derivatives of the broad-host-range multicopy plasmid pKT230 (17). As described under "Experimental Procedures," the derivatives used, pKTF1.2 and pKTR1.2, both contained the fpr gene and differed only in the orientation of the A. vinelandii DNA insert relative to the pKT230 vector. Fig. 8 shows the SDS-gel electrophoresis separation of cellfree extracts of wild-type A. vinelandii compared to the overproduction strains trans/pKTR1.2 and trans/pKTF1.2. Clearly there has been an overproduction of the NADPH:ferredoxin reductase in both trans/pKTF1.2 and trans/pKTR1.2. The fpr gene product overexpressed in A. vinelandii is indistinguishable in terms of activity and crystallization properties from the protein purified from A. vinelandii strain LM100. However, the amount of protein is much greater for the strain that has the gene in the same orientation as the kanamycin promoter on the vector. This observation is in contrast to the result obtained for the overexpression of FdI using the same pKT230 system (17). In that case, the protein was being maximally expressed from its own promoter such that the levels of overexpression were the same regardless of the orientation of the insert. The data in Fig. 8 show that something is preventing the maximal expression of the NADPH:ferredoxin reductase from its own promoter in trans/pKTR1.2. These data therefore suggest that unlike the situation with respect to FdI, expression from the fpr promotor may be tightly regulated, by a factor(s) whose concentration is limited.
Function and Regulation of the fpr Gene Product in E. coli and A. vinelandii-Investigators working on three very different problems have now converged on the same protein. First, the fpr gene product from E. coli was identified as a component of the anaerobic ribonucleotide reductase system (19). In that context, the NADPH:ferredoxin reductase mediates electron transfer from NADPH to Fld which then serves as a specific electron donor for the activation of anaerobic ribonucleotide reductase (24) and other enzymes involved in anaerobic metabolism (e.g. pyruvate formate-lyase (25,26)). Although A. vinelandii is an obligate aerobe, while fixing-N 2 it appears to maintain a very low internal O 2 concentration in order to protect the O 2 -sensitive enzyme nitrogenase (e.g. Ref. 28). Thus, the fpr gene product could serve a similar anaerobic enzyme activation function by reducing Fld in A. vinelandii. In A. vinelandii, however, Fld has an additional function as the immediate electron donor to nitrogenase, and, consequently, it is up-regulated under N 2 -fixing conditions (22,23). Our observation that the fpr product is also up-regulated under N 2 -fixing conditions (Fig. 7) suggests that in A. vinelandii the NADPH: ferredoxin reductase reported here may also serve as an electron donor to Fld in support of N 2 reduction.
Second, in our work, we encountered the fpr gene product while working on AvFdI. In that context, we have shown that the NADPH:ferredoxin reductase binds specifically to AvFdI and that it can mediate electron transfer between NADPH and FdI (14). In E. coli, the fpr gene product also mediates electron transfer between NADPH and ferredoxin (26). However, it should be noted that the only ferredoxin that has so far been isolated from E. coli is a [2Fe-2S] protein (32) that is very different from the 7Fe-containing AvFdI (1)(2)(3)(4)(5). Given the extent of similarity of the E. coli and A. vinelandii fpr gene products throughout their entire length (Fig. 4), the specificity of the A. vinelandii fpr product for FdI (14), and the known multiplicity of different types of ferredoxins in a number of organisms, it may be that one natural redox partner of NAD-PH:ferredoxin reductase in E. coli is an as yet unrecognized FdI-type ferredoxin.
The final way in which this protein was identified is through studies of oxygen toxicity in E. coli. In that case, mutants sensitive to methyl viologen, a superoxide radical propagator, were isolated, and the gene involved was cloned (33) and shown to be identical with the E. coli fpr gene (19). Subsequently, Fridovich and co-workers (34) showed that the gene product, NADPH:ferredoxin reductase, was part of an O 2 protection system that was controlled by the SoxRS regulon. SoxR is proposed to be an [Fe-S] protein (35). In response to superoxide it is converted into an activator of the soxS gene, and SoxS then activates the transcription of a number of other proteins including the fpr gene product. Fridovich and co-workers (34) proposed that reduced ferredoxins or flavodoxins might control the system by reducing SoxR which would then turn off the activation. Thus, it is proposed that when SoxR is oxidized, the soxS gene is activated causing NADPH:ferredoxin reductase to be overexpressed. It then reduces flavodoxin and ferredoxin, which in turn reduce SoxR, which then deactivates the SoxRS system (34,35). It is important to note that in A. vinelandii the fpr gene product initially came to our attention because it was overexpressed in strains that do not synthesize FdI (11,12). It is not overexpressed in strains that do not synthesize Fld (12). Further studies will therefore be directed at determining how FdI exerts its regulatory effect on fpr and examining the possibility that it might act through a SoxRS-like regulon in A. vinelandii.