Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2

False discovery and false nondiscovery rates in single-step multiple testing procedures

Results on the false discovery rate (FDR) and the false nondiscovery rate (FNR) are developed for single-step multiple testing procedures. In addition to verifying desirable properties of FDR and FNR as measures of error rates, these results extend previously known results, providing further insights, particularly under dependence, into the notions of FDR and FNR and related measures. First, considering fixed configurations of true and false null hypotheses, inequalities are obtained to explain how an FDR- or FNR-controlling single-step procedure, such as a Bonferroni or \u{S}id\'{a}k procedure, can potentially be improved. Two families of procedures are then constructed, one that modifies the FDR-controlling and the other that modifies the FNR-controlling \u{S}id\'{a}k procedure. These are proved to control FDR or FNR under independence less conservatively than the corresponding families that modify the FDR- or FNR-controlling Bonferroni procedure. Results of numerical investigations of the performance of the modified \u{S}id\'{a}k FDR procedure over its competitors are presented. Second, considering a mixture model where different configurations of true and false null hypotheses are assumed to have certain probabilities, results are also derived that extend some of Storey's work to the dependence case.Comment: Published at http://dx.doi.org/10.1214/009053605000000778 in the Annals of Statistics (http://www.imstat.org/aos/) by the Institute of Mathematical Statistics (http://www.imstat.org

The small FNR regulon of Neisseria gonorrhoeae: comparison with the larger Escherichia coli FNR regulon and interaction with the NarQ-NarP regulon

BACKGROUND: Neisseria gonorrhoeae can survive during oxygen starvation by reducing nitrite to nitrous oxide catalysed by the nitrite and nitric oxide reductases, AniA and NorB. The oxygen-sensing transcription factor, FNR, is essential for transcription activation at the aniA promoter, and full activation also requires the two-component regulatory system, NarQ-NarP, and the presence of nitrite. The only other gene known to be activated by the gonococcal FNR is ccp encoding a cytochrome c peroxidase, and no FNR-repressed genes have been reported in the gonococcus. In contrast, FNR acts as both an activator and repressor involved in the control of more than 100 operons in E. coli regulating major changes in the adaptation from aerobic to anaerobic conditions. In this study we have performed a microarray-led investigation of the FNR-mediated responses in N. gonorrhoeae to determine the physiological similarities and differences in the role of FNR in cellular regulation in this species. RESULTS: Microarray experiments show that N. gonorrhoeae FNR controls a much smaller regulon than its E. coli counterpart; it activates transcription of aniA and thirteen other genes, and represses transcription of six genes that include dnrN and norB. Having previously shown that a single amino acid substitution is sufficient to enable the gonococcal FNR to complement an E. coli fnr mutation, we investigated whether the gonococcal NarQ-NarP can substitute for E. coli NarX-NarL or NarQ-NarP. A plasmid expressing gonococcal narQ-narP was unable to complement E. coli narQP or narXL mutants, and was insensitive to nitrate or nitrite. Mutations that progressively changed the periplasmic nitrate sensing region, the P box, of E. coli NarQ to the sequence of the corresponding region of gonococcal NarQ resulted in loss of transcription activation in response to the availability of either nitrate or nitrite. However, the previously reported ligand-insensitive ability of gonococcal NarQ, the "locked on" phenotype, to activate either E. coli NarL or NarP was confirmed. CONCLUSION: Despite the sequence similarities between transcription activators of E. coli and N. gonorrhoeae, these results emphasise the fundamental differences in transcription regulation between these two types of pathogenic bacteria

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Fumarate and nitrate reduction (FNR) dependent activation of the Escherichia coli anaerobic ribonucleotide reductase nrdDG promoter

The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (fumarate and nitrate reduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR* demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations

Fumarate and nitrate reduction (FNR) dependent activation of the Escherichia coli anaerobic ribonucleotide reductase nrdDG promoter

The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (fumarate and nitrate reduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR* demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations. [Int Microbiol 2008; 11(1):49-56