Search PubMed⌕ Search

Biomedical subjects

Patricia J Kiley

Publications and source records attributed to Patricia J Kiley.

11 recordsLinked to original sources
PubMed2025

The Role of the [2Fe-2S] Cluster of Escherichia coli IscR in Responding to Redox-Cycling Agents.

The mechanisms by which cells respond to growth inhibitory redox-cycling agents is only partially understood. In Escherichia coli K12, the IscR regulon, which includes the ISC and SUF Fe-S cluster biogenesis machineries, is differentially expressed in response to these agents. Here, we report how one redox-cycling agent, phenazine methosulfate (PMS), regulates IscR activity via its [2Fe-2S] cluster cofactor. A direct role for IscR in mediating the response to PMS was inferred from the PMS-dependent weakening of [2Fe-2S]-IscR binding to an isc operon type 1 DNA site in vitro. This decrease in DNA binding was attributed to the accompanying oxidation of its [2Fe-2S]1+ cluster. Exposure of anaerobic cultures to PMS leads to increased isc expression, as expected from IscR cluster oxidation and impaired binding to type 1 sites in the isc promoter. However, this same anaerobic PMS treatment did not change expression of type 2 site promoters, such as suf, which require IscR that lacks an Fe-S cluster (apo-IscR) for effective transcriptional regulation. In contrast, PMS exposure under aerobic conditions significantly increased both isc and suf expression, indicating the formation of both [2Fe-2S]2+-IscR and apo-IscR. This effect was partially attributed to superoxide generation by PMS under aerobic conditions, as evidenced by a superoxide dismutase-deficient mutant showing a modest impact on isc and suf expression. Together, these findings provide new insights into redox-cycling dependent regulation of IscR activity and highlight the distinct activities of apo-IscR, [2Fe-2S]2+-IscR and [2Fe-2S]1+-IscR in controlling the IscR regulon.

Escherichia coli Proteins↗
PubMed2006

IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli.

IscR is an iron-sulphur (Fe-S) cluster-containing transcription factor that represses transcription of the operon containing its own gene and the iscSUA-hscBA-fdx genes, whose products are involved in Fe-S cluster biogenesis. In this study, global transcriptional profiling of Escherichia coli IscR(+) and IscR(-) strains grown under aerobic and anaerobic conditions indicated that 40 genes in 20 predicted operons were regulated by IscR. DNase I footprinting and/or in vitro transcription reactions identified seven new promoters under direct IscR control. Among these were genes encoding known or proposed functions in Fe-S cluster biogenesis (sufABCDSE, yadR and yhgI) and Fe-S cluster-containing anaerobic respiratory enzymes (hyaABCDEF, hybOABCDEFG and napFDAGHBC). The finding that IscR repressed expression of the hyaA, hybO and napF promoters specifically under aerobic growth conditions suggests a new mechanism to explain their upregulation under anaerobic growth conditions. Phylogenetic footprinting of the DNase I protected regions of seven promoters implies that there are at least two different classes of IscR binding sites conserved among many bacteria. The findings presented here indicate a more general role of IscR in the regulation of Fe-S cluster biogenesis and that IscR contributes to the O(2) regulation of several promoters controlling the expression of anaerobic Fe-S proteins.

Anaerobiosis↗
PubMed2005

ClpXP-dependent proteolysis of FNR upon loss of its O2-sensing [4Fe-4S] cluster.

The global regulator FNR from Escherichia coli controls the transcription of genes required for an anaerobic lifestyle. While previous studies have demonstrated that FNR activity is regulated by O2 through loss of dimerization upon destruction of its [4Fe-4S]2+ cluster, the present study reveals that monomeric FNR protein is also a target of proteolysis. We have found that turnover of FNR protein is increased selectively under aerobic growth conditions, when FNR is not active as a transcription factor and is primarily a metal-free, monomeric form (apo-FNR). This degradation of monomeric FNR was dependent on the ClpXP protease and required the presence of two amino acid sequences within FNR that resemble known ClpX recognition motifs. By measuring the turnover rates of various FNR mutants that have unique properties with respect to dimerization and Fe-S cluster stability, we have shown that loss of dimerization upon [4Fe-4S]2+ cluster destruction by O2 targets FNR for degradation by the ClpXP protease. In addition, by measuring the differential rate of FNR degradation upon switching aerobic cultures to anaerobic growth conditions, we provide evidence that pre-existing FNR apo-protein can be converted to [4Fe-4S]2+ -FNR. Finally, we address the physiological significance of FNR proteolysis by demonstrating that varying FNR protein levels over a small range under aerobic growth conditions has a direct effect on the function of FNR in O2 sensing.

Aerobiosis↗
PubMed2005

Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function.

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Deltafnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.

Aerobiosis↗
PubMed2005

Additional determinants within Escherichia coli FNR activating region 1 and RNA polymerase alpha subunit required for transcription activation.

The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(-61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the alpha subunit (alphaCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that alphaCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of alphaCTD required for FNR activation is more extensive than previously observed.

Alanine↗
PubMed2004

Superoxide destroys the [2Fe-2S]2+ cluster of FNR from Escherichia coli.

The oxygen sensing ability of the transcription factor FNR depends on the presence of a [4Fe-4S]2+ cluster. In the presence of O2, conversion of the [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster inactivates FNR, but the fate of the [2Fe-2S]2+ cluster in cells grown under aerobic conditions is unknown. The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the [2Fe-2S]2+ cluster, like the [4Fe-4S]2+ cluster, is not stable under these conditions. By quantifying the amount of [2Fe-2S]2+ cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the [2Fe-2S]2+ cluster. Mössbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the [2Fe-2S]2+ cluster. We conclude that the [2Fe-2S]2+ cluster of FNR is labile to superoxide both in vitro and in vivo. This lability may explain the absence of the [2Fe-2S]2+ cluster form of FNR under aerobic growth conditions.

Aerobiosis↗
PubMed2004

Kinetic analysis of the oxidative conversion of the [4Fe-4S]2+ cluster of FNR to a [2Fe-2S]2+ Cluster.

The ability of FNR to sense and respond to cellular O(2) levels depends on its [4Fe-4S](2+) cluster. In the presence of O(2), 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 approximately 2 Fe(2+) ions are released from the reaction of O(2) with the [4Fe-4S](2+) cluster. Fe(2+) 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 O(2)-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 O(2); a comparison of the in vitro and in vivo rates of conversion suggests that O(2)-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.

DNA↗
PubMed2003

Techniques for studying the oxygen-sensitive transcription factor FNR from Escherichia coli.

A large variety of techniques can be adapted for use with oxygen-sensitive samples. The growth of cells and in vivo analyses, as well as protein purification and in vitro assays, can be executed either by performing necessary steps in anaerobic environments (ranging from simple closed containers to the anaerobic chamber) or by circumventing the need for anaerobiosis with the use of oxygen-resistant protein variants.

Biochemistry↗
PubMed2003

The role of Fe-S proteins in sensing and regulation in bacteria.

Fe-S clusters are key to the sensing and transcription functions of three transcription factors, FNR, IscR and SoxR. All three proteins were discovered in Escherichia coli but experimental data and bioinformatic predictions suggest that homologs of these proteins exist in other bacterial species, highlighting the widespread nature of Fe-S-dependent regulatory networks. In addition, the nearly ubiquitous citric acid cycle enzyme, aconitase, plays a role in translational regulation in E. coli and Bacillus subtilis when it loses its Fe-S cluster. Although these regulatory proteins have the common feature of containing an Fe-S cluster, they differ in the physiological signals that they respond to. Therefore, these regulatory factors provide insights into the chemical versatility of Fe-S clusters.

Aconitate Hydratase↗
PubMed2002

Characterization of activating region 3 from Escherichia coli FNR.

Transcription activation of anaerobically induced genes in Escherichia coli is mediated through the action of the global anaerobic regulator FNR. Although regions of FNR involved in FNR-dependent transcription activation have been identified, the side-chains critical to the function of these regions are not known. In this study, alanine-scanning of amino acid residues 80-89 of FNR-activating region 3 (FNR-AR3) was used to determine which amino acid side-chains are required for transcription activation of class II FNR-dependent promoters. In vivo beta-galactosidase assays and in vitro transcription activation assays showed that Ala substitution of Ile81, Gly85 and Asp86 had the largest transcription activation defects, while comparison of the activity of single and double mutants indicated that Thr82, Glu83, Glu87 and Gln88 may contribute in a minor way to FNR-AR3 function. Site-directed mutagenesis of positions 81 and 86 showed that the hydrophobicity of Ile81 and the negative charge of Asp86 were important to FNR-AR3's function. Lastly, substitution of residues of E. coli FNR-AR3 with those more basic residues found in a subset of FNR homologs, such as Rhodobacter sphaeroides FnrL, resulted in a mutant strain that was unable to activate transcription from E. coli class II FNR-dependent promoters. In conclusion, this study demonstrates a requirement for negatively charged and hydrophobic side-chain residues in E. coli FNR-AR3 function, although there is likely to be some variability in the characteristics of this region in other members of the FNR family.

Amino Acid Sequence↗