Prediction of the Tertiary Structure and the Dimerization Interface of FNR Protein in Klebsiella pneumoniae

FNR (fumarate nitrate reduction regulator) is an important oxygen regulation protein, which plays a major role in altering gene expression between aerobic and anaerobic conditions. In this study, the tertiary structure of FNR protein in Klebsiella pneumoniae was established using homology modelling. The model was modified and optimized using molecular mechanics and molecular dynamics simulations. In addition, Support Vector Machines (SVMs) was applied to predict the possible binding sites patches of FNR. The residues conservations were further analyzed and it was found that the Asn138-Ser162 region of FNR protein was the putative dimerization interface.

Competition between NarL-dependent activation and Fis-dependent repression controls expression from the Escherichia coli yeaR and ogt promoters

The Escherichia coli NarL protein is a global gene regulatory factor that activates transcription at many target promoters in response to nitrate and nitrite ions. Although most NarL-dependent promoters are also co-dependent on a second transcription factor, FNR protein, two targets, the yeaR and ogt promoters, are activated by NarL alone with no involvement of FNR. Biochemical and genetic studies presented here show that activation of the yeaR promoter is dependent on the binding of NarL to a single target centred at position −43.5, whereas activation at the ogt promoter requires NarL binding to tandem DNA targets centred at position −45.5 and −78.5. NarL-dependent activation at both the yeaR and ogt promoters is decreased in rich medium and this depends on Fis, a nucleoid-associated protein. DNase I footprinting studies identified Fis-binding sites that overlap the yeaR promoter NarL site at position −43.5, and the ogt promoter NarL site at position −78.5, and suggest that Fis represses both promoters by displacing NarL. The ogt gene encodes an O6-alkylguanine DNA alkyltransferase and, hence, this is the first report of expression of a DNA repair function being controlled by nitrate ions.

Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR

ABSTRACT The role of the N-terminal region of the transcription factor FNR, which immediately precedes the first ligand (Cys20) of the [4Fe-4S] cluster, was investigated. We found that truncation mutants that removed residues 2 to 16 and 2 to 17 had wild-type levels of FNR protein but surprisingly altered O2 regulation.

Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12

ABSTRACT The facultative aerobe Escherichia coli K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the yeaR-yoaG operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that yeaR-yoaG operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that yeaR-yoaG operon transcription is activated by phospho-NarL protein independent of the Fnr protein. The phospho-NarL protein binding site is centered at position −43.5 with respect to the transcription initiation site. Expression from the Shewanella oneidensis MR-1 nnrS gene promoter, cloned into E. coli, similarly was activated by phospho-NarL protein independent of the Fnr protein. Recently, yeaR-yoaG operon transcription was shown to be regulated by the nitric oxide-responsive NsrR repressor (N. Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses reveal the individual contributions of the Nar and NsrR regulators to overall yeaR-yoaG operon expression and document the NsrR operator centered at position −32. Thus, control of yeaR-yoaG operon transcription provides an example of overlapping regulation by nitrate and nitrite, acting through the Nar regulatory system, and nitric oxide, acting through the NsrR repressor.

In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

FNR proteins are transcription regulators that sense changes in oxygen availability via assembly–disassembly of [4Fe–4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe–4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron–sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions, ∼45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR- and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis.

Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

An FNR-Type Regulator Controls the Anaerobic Expression of Hyn Hydrogenase in Thiocapsa roseopersicina

ABSTRACT The purple sulfur photosynthetic bacterium Thiocapsa roseopersicina BBS contains a heat-stable membrane-associated hydrogenase encoded by the hyn operon. Expression from the hyn operon regulatory region is up-regulated under anaerobic conditions. cis elements were mapped between positions −602 and −514 upstream from the hynS gene. Within this region two sequences that resemble DNA sites for FNR were recognized. The gene of an FNR homologue, FnrT, was identified in the genome of T. roseopersicina, and an fnrT knockout mutant was constructed. Anaerobic induction of hynS expression was abolished in the fnrT mutant, suggesting that FnrT is an activator of the hynS promoter. The T. roseopersicina hynS promoter could be activated in Escherichia coli, and this regulation was dependent on E. coli FNR. In vitro experiments with purified E. coli Ala154 FNR protein and purified E. coli RNA polymerase showed that FNR bound to two sites in the hyn regulatory region, that FNR could activate transcription initiation at the hynS promoter, and that FNR bound at the two target sites activated to different extents.

Kinetic Analysis of the Oxidative Conversion of the [4Fe-4S]2+ Cluster of FNR to a [2Fe-2S]2+ Cluster

ABSTRACT The ability of FNR to sense and respond to cellular O2 levels depends on its [4Fe-4S]2+ cluster. In the presence of O2, the [4Fe-4S]2+ cluster is converted to a [2Fe-2S]2+ cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that ∼2 Fe2+ ions are released from the reaction of O2 with the [4Fe-4S]2+ cluster. Fe2+ release was then used as an assay of reaction progress to investigate the rate of [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion in vitro. We also found that there was no detectable difference in the rate of O2-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O2; a comparison of the in vitro and in vivo rates of conversion suggests that O2-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.