F. Wayne Outten

A suf operon requirement for Fe–S cluster assembly during iron starvation in Escherichia coli

The suf and isc operons of Escherichia coli have been implicated in Fe–S cluster assembly. However, it has been unclear why E. coli has two systems for Fe–S cluster biosynthesis. We have examined the regulatory characteristics and mutant phenotypes of both operons to discern if the two operons have redundant functions or if their cellular roles are divergent. Both operons are similarly induced by hydrogen peroxide and the iron chelator 2,2′‐dipyridyl, although by different mechanisms. Regulation of the isc operon is mediated by IscR, whereas the suf operon requires OxyR and IHF for the response to oxidative stress and Fur for induction by iron starvation. Simultaneous deletion of iscS and most suf genes is synthetically lethal. However, although the suf and isc operons have overlapping functions, they act as distinct complexes because the SufS desulphurase alone cannot substitute for the IscS enzyme. In addition, suf deletion mutants are more sensitive to iron starvation than isc mutants, and the activity of the Fe–S enzyme gluconate dehydratase is diminished in the suf mutant during iron starvation. These findings are consistent with the model that the isc operon encodes the housekeeping Fe–S cluster assembly system in E. coli, whereas the suf operon is specifically adapted to synthesize Fe–S clusters when iron or sulphur metabolism is disrupted by iron starvation or oxidative stress.

Transcriptional Activation of an Escherichia coli Copper Efflux Regulon by the Chromosomal MerR Homologue, CueR

Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA andyacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbIas cueR for the Cu effluxregulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response.

Fe-S Cluster Assembly Pathways in Bacteria

SUMMARY Iron-sulfur (Fe-S) clusters are required for critical biochemical pathways, including respiration, photosynthesis, and nitrogen fixation. Assembly of these iron cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. Multiple Fe-S cluster assembly pathways are present in bacteria to carry out basal cluster assembly, stress-responsive cluster assembly, and enzyme-specific cluster assembly. Although biochemical and genetic characterization is providing a partial picture of in vivo Fe-S cluster assembly, a number of mechanistic questions remain unanswered. Furthermore, new factors involved in Fe-S cluster assembly and repair have recently been identified and are expanding the complexity of current models. Here we attempt to summarize recent advances and to highlight new avenues of research in the field of Fe-S cluster assembly.

DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in Escherichia coli.

MerR-like DNA distortion mechanisms have been proposed for a variety of stress-responsive transcription factors. TheEscherichia coli ZntR protein, a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation ofzntA, a gene involved in Zn(II) detoxification. To determine whether the MerR DNA distortion mechanism is conserved among MerR family members, we have purified ZntR to homogeneity and shown that it is a zinc receptor that is necessary and sufficient to stimulate Zn-responsive transcription at the zntA promoter. Biochemical, DNA footprinting, and in vitro transcription assays indicate that apo-ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to apo-MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. We propose that this zinc-sensing homologue of MerR restructures the target promoter in a manner similar to that of other stress-responsive transcription factors. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal but as yet unknown concentration.

SufE Transfers Sulfur from SufS to SufB for Iron-Sulfur Cluster Assembly

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.

IscR Controls Iron-Dependent Biofilm Formation in Escherichia coli by Regulating Type I Fimbria Expression

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.

Native Escherichia coli SufA, Coexpressed with SufBCDSE, Purifies as a [2Fe−2S] Protein and Acts as an Fe−S Transporter to Fe−S Target Enzymes

Iron-sulfur (Fe-S) clusters are versatile biological cofactors that require biosynthetic systems in vivo to be assembled. In Escherichia coli, the Isc (iscRSUA-hscBA-fdx-iscX) and Suf (sufABCDSE) pathways fulfill this function. Despite extensive biochemical and genetic analysis of these two pathways, the physiological function of the A-type proteins of each pathway (IscA and SufA) is still unclear. Studies conducted in vitro suggest two possible functions for A-type proteins, as Fe-S scaffold/transfer proteins or as iron donors during cluster assembly. To resolve this issue, SufA was coexpressed in vivo with its cognate partner proteins from the suf operon, SufBCDSE. Native SufA purified anaerobically using this approach was unambiguously demonstrated to be a [2Fe-2S] protein by biochemical analysis and UV-vis, Mossbauer, resonance Raman, and EPR spectroscopy. Furthermore, native [2Fe-2S] SufA can transfer its Fe-S cluster to both [2Fe-2S] and [4Fe-4S] apoproteins. These results clearly show that A-type proteins form Fe-S clusters in vivo and are competent to function as Fe-S transfer proteins as purified. This study resolves the contradictory results from previous in vitro studies and demonstrates the critical importance of providing in vivo partner proteins during protein overexpression to allow correct biochemical maturation of metalloproteins.

SufD and SufC ATPase activity are required for iron acquisition during in vivo Fe-S cluster formation on SufB

In vivo biogenesis of Fe-S cluster cofactors requires complex biosynthetic machinery to limit release of iron and sulfide, to protect the Fe-S cluster from oxidation, and to target the Fe-S cluster to the correct apoenzyme. The SufABCDSE pathway for Fe-S cluster assembly in Escherichia coli accomplishes these tasks under iron starvation and oxidative stress conditions that disrupt Fe-S cluster metabolism. Although SufB, SufC, and SufD are all required for in vivo Suf function, their exact roles are unclear. Here we show that SufB, SufC, and SufD, coexpressed with the SufS-SufE sulfur transfer pair, purify as two distinct complexes (SufBC(2)D and SufB(2)C(2)) that contain Fe-S clusters and FADH(2). These studies also show that SufC and SufD are required for in vivo Fe-S cluster formation on SufB. Furthermore, while SufD is dispensable for in vivo sulfur transfer, it is absolutely required for in vivo iron acquisition. Finally, we demonstrate for the first time that the ATPase activity of SufC is necessary for in vivo iron acquisition during Fe-S cluster assembly.

The SufBCD Fe-S Scaffold Complex Interacts with SufA for Fe-S Cluster Transfer

Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly transpires via multiprotein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The Gram-negative bacterium Escherichia coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.

The Impact of O2 on the Fe–S Cluster Biogenesis Requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the 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 by twofold to threefold 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, FNR-L28H activity was more impaired under aerobic conditions than under 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 under aerobic and anaerobic conditions, as shown by (55)Fe-labeling 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 into [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.