Transcription activation at Escherichia coli FNR-dependent promoters by the gonococcal FNR protein: effects of a novel S18F substitution and comparisons with the corresponding substitution in E. coli FNR

Abstract

The Neisseria gonorrhoeae genome encodes a homologue of the Escherichia coli FNR protein (the fumarate and nitrate reductase regulator). Despite its similarity to E. coli FNR, the gonococcal FNR only partially complemented an E. coli fnr mutation. After error-prone PCR mutagenesis of the gonococcal fnr gene, we identified four mutant fnr derivatives carrying the same S18F substitution, and we showed that the mutant FNR could activate transcription from a range of class I and class II FNR-dependent promoters in E. coli. Prompted by the similarities between gonococcal and E. coli FNR, we made changes in gonococcal fnr that created substitutions that are equivalent to previously characterized substitutions in E. coli FNR. First, our experiments showed that cysteine, C116, in the gonococcal FNR, equivalent to C122 in E. coli FNR, is essential, presumably because, as in E. coli FNR, it binds to an iron-sulfur center. Second, the L22H and D148A substitutions in gonococcal FNR were made. These changes are equivalent to the L28H and D154A changes in E. coli FNR, which had been shown to increase FNR activity in the presence of oxygen. We show that the effects of these substitutions in gonococcal FNR are distinct from those of the S18F substitution. Similarly, substitutions in the putative activating regions of gonococcal FNR were made. We show that the activity of gonococcal FNR in E. coli can be increased by transplanting certain activating regions from E. coli FNR. The effects of these substitutions are additive to those due to S18F. From these data, we conclude that the effects of the S18F substitution in gonococcal FNR are distinct from the effects of the other substitutions. S18 is immediately adjacent to one of three N-terminal cysteine residues that coordinate the iron-sulfur center, and thus the S18F substitution is most likely to stabilize this center. Support for this came from complementary experiments in which we created the S24F substitution in E. coli FNR, which is equivalent to the S18F substitution in gonococcal FNR. Our results show that the S24F substitution changes the activity of E. coli FNR and that the changes are distinct from those due to previously characterized substitutions.

FIG. 1.

Structure of N. gonorrhoeae and E. coli FNR proteins. (A) The structure of E. coli FNR protein is modeled on the crystal structure of the related E. coli CRP protein. Alpha-helices are lettered A to F; beta-sheets are numbered 1 to 12. The four cysteine residues coordinating the iron-sulfur center and portions of the ARs altered in Fig. 7 are labeled. (B) The amino acid sequences of the gonococcal (Ng) and E. coli (Ec) FNR proteins were aligned. A vertical line denotes residue identity, a colon denotes strong residue similarity, and a dot denotes weak residue similarity. Residues comprising AR1 of E. coli FNR are marked with an asterisk, residues comprising AR2 are marked with a caret, and residues comprising AR3 are marked with a plus sign. Alpha-helices are lettered A to F and marked with rectangles; beta-sheets are numbered 1 to 12 and marked with arrows. The four cysteine residues that coordinate the iron-sulfur center of E. coli FNR are underlined.

FIG. 2.

Western blotting was used to detect the expression of E. coli and N. gonorrhoeae FNR proteins. E. coli cultures were grown to late exponential phase; N. gonorrhoeae cultures were grown microaerobically. Lanes were loaded with equal biomass as follows: lane 1, E. coli JRG1728 transformed with pGCFNR3 carrying an S18F substitution; lane 2, E. coli JRG1728 transformed with pGCFNR3; lane 3, N. gonorrhoeae RUG7022; lane 4, N. gonorrhoeae RUG7001; lane 5, E. coli JRG1728 transformed with pFNR; lane 6, E. coli JRG1728 transformed with pGCFNR3 carrying an L22H substitution. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membrane. FNR protein was detected using antisera raised against E. coli FNR as a primary antibody and an alkaline phosphatase-conjugated secondary antibody. A band corresponding to FNR protein is present in lanes containing protein from E. coli transformed with plasmids expressing E. coli or gonococcal FNR proteins, and in N. gonorrhoeae strain RUG7001. No band is present in lane 3, which contains proteins from N. gonorrhoeae fnr mutant strain RUG7022.

FIG. 3.

Effects of the substitutions L22H and D148A in the presence or absence of the S18F substitution on transcription activation by the gonococcal FNR at class I and class II FNR-dependent promoters. Mutations were introduced into the gonococcal fnr gene on plasmid pGCFNR3 using the QuikChange method. The mutated plasmids were transformed into strain JRG1728 carrying the consensus FNR-dependent promoter-lacZ fusions FF(−41.5) (A) or FF(−71.5) (B) integrated into the phage λ attachment site. Each transformant was assayed in duplicate from two independent cultures; units of activity are nanomoles of ONPG hydrolyzed · minute⁻¹ · (milligram of bacteria [dry mass])⁻¹. Error bars, standard deviations.

FIG. 4.

Effect on transcription activation at a class II consensus FNR-dependent promoter of replacing serine 18 of the gonococcal FNR in plasmid pGCFNR3 with different amino acids. The gonococcal fnr genes carried on pGCFNR3 were mutated using the QuikChange method and the resulting plasmids transformed into E. coli strain JRG1728λFF(−41.5), which carries a chromosomal consensus FNR-binding site centered between 41 and 42 bases upstream from lacZ integrated into the phage λ attachment site (20). Purified transformants, together with pFNR expressing the E. coli FNR as a positive control, were grown aerobically (open bars) and anaerobically (filled bars) in LB-glucose and assayed for β-galactosidase activity. Other details are as explained in the legend to Fig. 3.

FIG. 5.

Identification of the central cysteine residue of the gonococcal FNR that is essential for transcription activation by FNR^Ng (S18F). The QuikChange method was used to change three cysteine residues on the central part of the gonococcal FNR to alanine, and the effects of the substitutions on transcription activation by FNR^Ng and FNR^Ng (S18F) in E. coli was determined. Each derivative gonococcal FNR was assayed in strain JRG1728 carrying the consensus FNR-dependent promoter-lacZ fusions FF(−41.5) (A) or FF(−71.5) (B) integrated into the phage λ attachment site. Open bars: aerobic cultures; hatched bars, anaerobic cultures. Other details are as explained in the legend to Fig. 3.

FIG. 6.

Gel retardation assays to study the effects of the S18F and S24F substitutions alone or in combination with D148A or D154A substitutions on binding of gonococcal or E. coli FNR to DNA in vitro. End labeled FF(−41.5) promoter DNA was incubated with various concentrations of FNR proteins. (A) The concentration of FNR protein in each reaction was as follows: lane 1, no protein; lanes 2, 6 and 10, 0.05 μM; lanes 3, 7 and 11, 0.1 μM; lanes 4, 8 and 12, 1 μM; and lanes 5, 9 and 13, 3 μM. (B) The concentration of FNR protein in each reaction mixture was as follows: lane 1, no protein; lane 2, 0.05 μM; lane 3, 0.1 μM; lane 4, 0.5 μM; lane 5, 1 μM; lane 6, 0.025 μM; lane 7, 0.05 μM; lane 8, 0.25 μM; lane 9, 0.5 μM; lane 10, 1.5 μM; lane 11, 2.1 μM.

FIG. 7.

Effects of AR substitutions on transcription activation by the gonococcal FNR at the FF(−41.5) promoter. Mutations detailed in Fig. 1 were introduced into the gonococcal fnr gene on plasmid pGCFNR3 using the QuikChange method. The mutated plasmids were transformed into strain JRG1728 carrying the consensus FNR-dependent promoter-lacZ fusion FF(−41.5) integrated into the phage λ attachment site. Open bars: aerobic cultures; hatched bars, anaerobic cultures. The upper horizontal line marks transcription activity due to E. coli FNR during anaerobic growth; the lower and middle horizontal lines mark transcription activity due to wild-type and S18F substituted gonococcal FNR during anaerobic growth, respectively. Other details are as explained in the legend to Fig. 3.

FIG. 8.

Effects of an S24F substitution, corresponding to S18F in the gonococcal FNR, on transcription activation by the E. coli FNR protein. Mutations were introduced into the E. coli fnr gene on plasmid pFNR using the QuikChange method. The mutated plasmids were transformed into strain JRG1728 carrying the consensus FNR-dependent promoter-lacZ fusions FF(−41.5) (A) or FF(−71.5) (B) integrated into the phage λ attachment site. The upper horizontal line marks transcription activity due to the unsubstituted E. coli FNR during anaerobic growth (hatched bars); the lower line shows the corresponding activity for the aerobic culture (open bars). Other details are as explained in the legend to Fig. 3.