SUMMARY Fe-S clusters are essential across the biological world, yet how cells regulate expression of Fe-S cluster biogenesis pathways to cope with changes in Fe-S cluster demand is not well understood. Here, we describe the mechanism by which IscR, a [2Fe-2S] cluster-containing regulator of Escherichia coli, adjusts the synthesis of the Isc Fe-S biogenesis pathway to maintain Fe-S homeostasis. Our data indicate that a negative feedback loop operates to repress transcription of the iscRSUA-hscBA-fdx operon, encoding IscR and the Isc machinery, through binding of [2Fe-2S]-IscR to two upstream binding sites. IscR was shown to require primarily the Isc pathway for synthesis of its Fe-S cluster, providing a link between IscR activity and demands for Fe-S clusters through the levels of the Isc system. Surprisingly, the isc operon was more repressed under anaerobic conditions, indicating increased Fe-S cluster occupancy of IscR and decreased Fe-S cluster biogenesis demand relative to aerobic conditions. Consistent with this notion, overexpression of a Fe-S protein under aerobic conditions, but not under anaerobic conditions, led to derepression of PiscR. Together, these data show how transcriptional control of iscRSUA-hscBA-fdx by [2Fe-2S]-IscR allows E. coli to respond efficiently to varying Fe-S demands.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

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By sensing and responding to environmental O 2 , facultative anaerobes are able to adopt the most energy-efficient metabolic processes for promoting cell growth under a variety of conditions. In Escherichia coli, the shift between aerobic and anaerobic metabolism, which is controlled primarily by the global transcriptional regulator FNR (13,23,32), is known to involve the repression and activation of hundreds of genes (25). However, the reactions that control the steps from O 2 sensing to changes in gene expression have not been fully described. The present study focuses on the O 2 -sensing mechanism of FNR and the rapidity with which FNR reacts with O 2 in order to adapt to shifting environmental conditions through appropriate changes in gene expression.Our current model for cellular O 2 sensing by FNR is based upon the critical discovery that FNR is directly inactivated by O 2 in vitro (12,15,16,19). FNR is a homodimeric DNA-binding protein containing approximately one 2ϩ cluster per subunit (called 4Fe-FNR) (3,16,19). The ability of FNR to function as a transcription factor depends on the integrity of a [4Fe-4S] 2ϩ cluster, which promotes a conformation that is necessary for FNR dimerization, site-specific DNA binding, and transcriptional regulation (12,16,19,21 cluster by superoxide, a by-product of aerobic metabolism (28). In addition, it is unknown whether the apo-FNR generated by the superoxide pathway is a substrate for the resynthesis of 4Fe-FNR in vivo. A previous study indicated that FNR present in aerobic cells can be activated upon a shift to anaerobic conditions in the presence of a protein synthesis inhibitor (8). However, it is unknown whether the FNR that was activated following the shift arose from apo-FNR, which was generated by cluster degradation, or from newly synthesized FNR, which had yet to acquire a Fe-S cluster. While it is still unresolved whether newly synthesized FNR protein is biochemically distinct from apo-FNR, a previous study has shown that the 2ϩ cluster can be assembled in vitro into FNR that has been purified (either aerobically or anaerobically) under conditions that produce clusterless FNR, suggesting that perhaps the apo-FNR form can be recycled to 4Fe-FNR in cells (32). An understanding of the rates by which all of these processes occur in cells is critical for developing a comprehensive model of O 2 sensing by FNR.As a first step toward addressing this question, we monitored the rate of conversion of 4Fe-FNR to 2Fe-FNR in vitro by measuring the release of iron ions in the reaction. The rate of inactivation of FNR by O 2 in cells has also been measured in order to analyze the relevance of the in vitro reaction to the in vivo process. Finally, we also examined the effect of the interaction of FNR with DNA on the rate of 4Fe-FNR to 2Fe-FNR conversion by O 2 . MATERIALS AND METHODSIsolation of 4Fe-FNR. 4Fe-FNR was isolated from anaerobically prepared cells in a Coy anaerobic chamber as previously described, by using a PolyCAT A

Iron-sulfur (Fe-S) clusters are fundamental to numerous biological processes in most organisms, but these protein cofactors can be prone to damage by various oxidants (e.g., O2, reactive oxygen species, and reactive nitrogen species) and toxic levels of certain metals (e.g., cobalt and copper). Furthermore, their synthesis can also be directly influenced by the level of available iron in the environment. Consequently, the cellular need for Fe-S cluster biogenesis varies with fluctuating growth conditions. To accommodate changes in Fe-S demand, microorganisms employ diverse regulatory strategies to tailor Fe-S cluster biogenesis according to their surroundings. Here, we review the mechanisms that regulate Fe-S cluster formation in bacteria, primarily focusing on control of the Isc and Suf Fe-S cluster biogenesis systems in the model bacterium Escherichia coli.

Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins.

Summary Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.

SummaryIn this study, the function of two established Fe-S cluster biogenesis pathways, Isc (Iron sulfur cluster) and Suf (Sulfur mobilization), was compared under aerobic and anaerobic growth conditions by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for activity of FNR under anaerobic conditions. Assaying expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon, revealed that under anaerobic conditions FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated 2-3 fold in Isc − strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O 2 -stable [4Fe-4S] cluster FNR variant, FNR-L28H, showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, activity of FNR-L28H was more impaired under aerobic conditions compared to anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H between aerobic and anaerobic conditions as shown by 55 Fe labelling experiments. Using 35 S-methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O 2 -inactivated apo-FNR to [4Fe-4S]-FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.