In Azotobacter vinelandii, the E1 subunit of the pyruvate dehydrogenase complex binds fpr promoter region DNA and ferredoxin I

Abstract

In Azotobacter vinelandii, deletion of the fdxA gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the fpr gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to fdxA deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the fpr gene. Here, we report the identification of the A. vinelandii protein that binds specifically to the paraquat activatable fpr promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix-turn-helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1.

Figure 1

A. vinelandii fprBP binds specifically to a sequence that is almost identical to the SoxS consensus sequence of E. coli. (A) The A. vinelandii fpr promoter region contains a SoxS-like sequence (referred to as S) (9) and a palindrome (underlined; referred to as P) (11). As described in Methods, a set of oligonucleotides was constructed with different regions mutated as indicated by dashed lines. (B) Gel mobility shift assays used partially purified fprBP (20 μg) and the oligonucleotides (1 fmol) indicated in A and methods described in ref. and the Methods section of this paper. The gels were run for 2 hr, such that the free probe ran off the gel and only the upper shifted band is shown. For full-length gels, see refs. and and Fig. 4.

Figure 2

Identification of fprBP for sequencing. (A) Silver-stained 7.5% SDS/polyacrylamide gel of partially purified fprBP where the gel was run until the 62-kDa band of the Biolabs (Northbrook, IL) protein markers reached the bottom. The molecular mass positions indicated here refer only to the gel in A (markers not shown). (B) Southwestern blot, run until the dye reached the bottom of the gel, and (S⁺P⁺) DNA. (C) UV crosslinking using 20 μg of protein and (S⁺P⁺) DNA. (D) The excised fprBP band for identification by matrix-assisted laser desorption ionization mass spectrometry. Lane a, Coomassie-stained gel; and lane b, Southwestern blot identifying fprBP as the upper band. See Methods for details and references.

Figure 3

Recombinant A. vinelandii PDHE1 is identical to fprBP. (A) Coomassie-stained 7.5% SDS/polyacrylamide gel of pure PDHE1. The molecular mass positions indicated here refer only to the gel in A (markers not shown). (B) Gel mobility shift assay with PDHE1 (50 pmol) and 1 fmol of the mutated fpr promoter region (SoxS⁻) (see Fig. 1) (lane 1); 1 fmol of the wild-type fpr promoter region (S⁺P⁺) (lane 2); and with addition of a 100× excess of unlabeled (S⁺P⁺) DNA to the sample (lane 3). For comparison, the position of the fprBP gel shift with (S⁺P⁺) using the partially purified fprBP is shown in lane 4, and in lane 5 with additional 100× excess of unlabeled (S⁺P⁺) DNA.

Figure 4

Titration of PDHE1. A total of 50 fmol of labeled 317-bp DNA was used per assay. Lanes: 1, DNA alone; 2–5, same as lane 1 plus increased quantities of pure PDHE1 (3.25 pmol, 6.5 pmol, 13 pmol, 19.5 pmol). Lower band is free probe; upper band is DNA/PDHE1 complex.

Figure 5

Sequence alignment of the putative helix–turn–helix motif region in A. vinelandii PDHE1 and P. aeruginosa PDHE1. : represents identical; . represents similar. The last line shows the predicted helix–turn–helix region in A. vinelandii PDHE1, using the nnpredict program by Kneller et al. (21) H, helix; –, turn.

Figure 6

Gel mobility shift assays show that PDHE1 interacts specifically with FdI. (A) fprBP (20 μg) is incubated with (S⁺P⁺) as a DNA probe alone (lane 1) and in the presence of: A. vinelandii FdI (lane 2); A. vinelandii apoFdI (lane 3); A. vinelandii flavodoxin I (lane 4); A. vinelandii flavodoxin II (lane 5); and spinach FdI (lane 6) (20 μg each). A. vinelandii FdIII (lane 2) was also tested and does not give a supershift. (B) Recombinant PDHE1 (50 pmol) is incubated with 1 fmol of (S⁺P⁺) DNA alone (lane 1) and in the presence of: FdI (800 pmol) (lane 2) and after addition of a 100× excess of the unlabeled (S⁺P⁺) DNA-probe to the FdI/PDHE1 sample (lane 3). (C) Gel shift with recombinant PDHE1 (50 pmol) incubated with 1 fmol of (S⁺P⁺) DNA in the presence of: A. vinelandii FdI (lane 1) and A. vinelandii FdI (1.6 nmol) and Av FPR (0.7 nmol) (lane 2). In A–C, a, DNA plus DNA-binding protein (fprBP or recombinant PDHE1); b, same as a plus FdI; c, same as b plus FPR. (D) Titration of FdI. A total of 50 fmol of labeled 317-bp DNA was used per assay. Lanes: 1, DNA alone; 2, same as lane 1 plus 10 pmol of pure PDHE1; 3, same as lane 2 plus 40 pmol of FdI; 4, same as lane 2 plus 80 pmol of FdI.