Global Iron-dependent Gene Regulation in Escherichia coli

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex. Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex. Iron is an essential minor element for most organisms, playing vital roles in many important biological processes including photosynthesis, N2 fixation, methanogenesis, H2 production, and consumption, respiration, the TCA 1The abbreviations used are: TCA, Tricarboxylic Acid Cycle; Tricine, N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine; Fur, ferric uptake regulation; EPR, electron paramagnetic resonance.1The abbreviations used are: TCA, Tricarboxylic Acid Cycle; Tricine, N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine; Fur, ferric uptake regulation; EPR, electron paramagnetic resonance. cycle, oxygen transport, and DNA biosynthesis. However, despite the indispensability of iron, it is also potentially toxic due to its tendency to catalyze free radical generation. In addition, the extremely poor solubility of the oxidized, ferric form leads to bioavailability problems (1Andrews S.C. Adv. Microb. Physiol. 1998; 40: 281-351Crossref PubMed Google Scholar). Organisms counter the difficulties posed by iron nutrition in a number of ways. One common mechanism involves the solubilization of extracellular iron, by reduction or chelation, followed by internalization via specific transporters. Another widespread approach is the deposition of intracellular iron stores within ferritin molecules that can be subsequently utilized to abrogate the effects of iron restriction (1Andrews S.C. Adv. Microb. Physiol. 1998; 40: 281-351Crossref PubMed Google Scholar, 2Guerinot M-L. Annu. Rev. Microbiol. 1994; 48: 743-772Crossref PubMed Scopus (524) Google Scholar). Iron metabolism in Escherichia coli K-12 is particularly well studied making it a model organism for investigations on iron-homeostatic processes. Like other bacteria, as well as fungi and some plants, it utilizes high-affinity extracellular ferric-chelators, called siderophores, to solubilize iron prior to transport (3Earhart C.F. Neidhardt F.C. Escherichia coli and Salmonella: Cellular 4: 172-177Crossref PubMed Scopus (566) Google Scholar). It also possesses three ferri-siderophore periplasmic-binding protein-dependent ABC-transporter systems, FecBCDE, FepBCDEFG, and FhuBCD, and, like many other bacteria, can take up ferrous iron anaerobically via FeoB. In addition, E. coli contains three iron storage proteins (Bfr, FtnA, and FtnB) of which FtnA plays the major storage role (5Abdul-Tehrani H. Hudson A.J. Chang Y-S. Timms A.R. Hawkins C. Williams J.M. Harrison P.M. Guest J.R. Andrews S.C. J. Bacteriol. 1999; 181: 1415-1428Crossref PubMed Google Scholar). Not surprisingly, the iron acquisition and storage systems are regulated in response to iron availability. This regulation is mediated by the homodimeric repressor protein, Fur, which employs ferrous iron as co-repressor (4Hantke K. Curr. Opin. Microbiol. 2001; 4: 172-177Crossref PubMed Scopus (566) Google Scholar). There is evidence that the Fe2+-Fur complex also represses genes (cyoA, flbB, fumC, gpmA, metH, nohB, purR, and sodA) involved in various non-iron functions (respiration, flagella chemotaxis, the TCA cycle, glycolysis, methionine biosynthesis, phage-DNA packaging, purine metabolism, and redox-stress resistance) so it can thus be considered to be a global regulator (6Stojiljkovic I. Bäumler A.J. Hantke K. J. Mol. Biol. 1994; 236: 531-545Crossref PubMed Scopus (308) Google Scholar, 7Park S.J. Gunsalus R.P. J. Bacteriol. 1995; 177: 6255-6262Crossref PubMed Scopus (124) Google Scholar, 8Vassinova N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar, 9Touati D. J. Bacteriol. 1988; 170: 2511-2520Crossref PubMed Google Scholar). Fe2+-Fur represses transcription by binding to a 19-bp sequence, designated the “iron box,” normally located near the Pribnow box of cognate promoters. Fur can also act as a transcriptional activator switching on genes encoding the iron-containing proteins aconitase A, Bfr, FtnA, fumarases A and B, succinate dehydrogenase, and superoxide dismutase B (7Park S.J. Gunsalus R.P. J. Bacteriol. 1995; 177: 6255-6262Crossref PubMed Scopus (124) Google Scholar, 10Tseng C-P. FEMS Microbiol. Lett. 1997; 157: 67-72Crossref PubMed Google Scholar, 11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar). This activation appears to be indirect and seems to involve (at least in some cases) the Fe2+-Fur repressed regulatory RNA, RyhB (11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar). Here we use transcriptional profiling to extend the Fur modulon of E. coli. Over 100 Fe2+-Fur-regulated genes were detected, most of which have not been previously reported. These include unknown genes potentially involved in iron acquisition. A large number of energy metabolism genes, mainly encoding Fe-containing respiratory complexes, were found to be Fe2+-Fur induced. This represents a major new functional category for inclusion within the Fur modulon. 55Fe-labeling studies and whole-cell spectroscopy showed that fur mutants are deficient in iron-containing proteins. Together, the data provide an explanation for the low iron contents of fur mutants and reveal a new Fur-dependent mechanism for iron homeostasis. Bacterial Strains and Culture Conditions—E. coli strains were grown in Luria-Bertani (L broth) medium (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY1989Google Scholar) at 37 °C and shaken at 250 rpm in an orbital shaker. Iron limitation was induced by inclusion of the ferrous iron chelator 2,2′-dipyridyl (dip) at 200 μm. RNA Isolation, Preparation of Radiolabeled cDNA, and Real-Time RT-PCR—Cultures of E. coli wild-type (MC4100), wild-type with dip and an E. coli fur mutant (H1941: MC4100 fur) were grown to an OD650 nm of 1.0 (six replicates for each condition). A 1-ml sample from each culture was harvested by centrifugation and total RNA extracted using the Qiagen RNeasy® kit. RNA was treated with RNase-free DNase I (Promega). Each set of six replicate samples was pooled into two groups of three to control for slight growth and extraction variations. Pooled total RNA samples were then used as templates for production of 33P-labeled cDNA using random hexaprimers (Promega), and the labeled cDNA probes purified using G25-Sephadex columns, as described by Sigma-Genosys. Quantitative RT-PCR was performed using an ABI 5700, the Sybr Green RT-PCR kit (Qiagen) and primers designed to amplify 50–80-bp fragments. Specificity was confirmed by electrophoretic analysis of the reaction products and by inclusion of or free RNA samples were from used for and cDNA was to two E. coli gene as described by the and were then to low for and the were then with a at were prior to as described by the were using for each were using were used to the which was then from the gene in with two were considered to of each of the within an array was by as of total This array were performed in and with coli and fur mutant fur) were grown in with or 200 to and were harvested by centrifugation and of were in of of were with and or were with were then at using a a of with a of nm U. N. S. FEMS Microbiol. Lett. 1998; Google Scholar). and cytochrome were as previously described PubMed Google Scholar, K. K. J. Biol. Scholar). to EPR were in in to from the of each sample was by the of followed by EPR were at on a EPR with an system. A was EPR samples were in EPR were as 100 were as previously described PubMed Scopus Google Scholar). of 55Fe-labeled E. coli coli were grown in of OD650 nm were harvested by centrifugation in the and in at were then by the J. Mol. Biol. PubMed Scopus Google Scholar) with the was used in of was was at the and was to a of were in and in of were then under and with of by transcriptional profiling was used to genes regulated by and Fur and transcription of MC4100 grown in was with of the wild-type grown with an chelator (dip) and the fur mutant fur) grown were harvested at an OD650 nm of to major growth were for the three conditions. 33P-labeled cDNA was from the RNA samples using and random and was to E. coli Each array was performed in using pooled RNA samples from three with of of the or wild-type fur mutant, and of the Fur modulon are regulated by iron and Fur in only genes that were regulated by dip and the fur are considered genes were found to be regulated by the Fe2+-Fur complex of which were repressed and induced. These genes into three major iron metabolism energy production and and Fur regulation of genes involved in iron and genes involved in energy and genes involved in energy iron- and iron- and genes Iron Iron most of the known iron-acquisition genes were induced by the chelator and the fur the enterobactin genes were the most genes to that energy is not on enterobactin production of iron In the uptake genes were that enterobactin production systems are by iron the acquisition previously the and genes involved in ferrous iron, B, and were repressed by the Fe2+-Fur as were the and genes for the outer However, the the ferric uptake was not by the chelator or fur it is known that transcriptional are Fe2+-Fur regulated K. Mol. PubMed Scopus Google Scholar). for is be to which probably functions in iron-sulfur cluster iron and J. Bacteriol. 2001; PubMed Scopus Google was also Fe2+-Fur repressed of as previously Hantke K. J. Bacteriol. 1999; 181: PubMed Google Scholar). However, the which genes with a role in iron-sulfur cluster was not Fe2+-Fur the gene encoding a to be involved in iron from was J.M. A.J. Andrews S.C. Guest J.R. PubMed Scopus Google Scholar) the gene the protein, ferritin A, was repressed by the chelator and fur as previously (5Abdul-Tehrani H. Hudson A.J. Chang Y-S. Timms A.R. Hawkins C. Williams J.M. Harrison P.M. Guest J.R. Andrews S.C. J. Bacteriol. 1999; 181: 1415-1428Crossref PubMed Google Scholar, 11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar). A from the of genes in I is for gene is known to be Fe2+-Fur its expression is and so is to the and S. C. the array data are generally with expression studies on the of iron array analysis the of several unknown genes with potential functions in iron acquisition These were of their and expression or by their with such genes. are into in of which the of genes and two proteins that are to each not to other E. coli a outer membrane previously to be involved in and K. FEMS Microbiol. Lett. a of a encoded by an gene to be involved in iron uptake Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and a of unknown that the is complex cluster of three genes a of the high-affinity ferrous iron of a potential of unknown and potentially of unknown A was previously in the of K. T. J. Bacteriol. 2001; PubMed Scopus Google and a is in the of This as for many other that these proteins groups prior to to the of the genes are found in the of at least 7 other that these genes form a functional to of a with the A. PubMed Scopus Google we that as a ferrous iron and that and act as a novel iron or is cluster of three genes, that to form an which is to ABC to outer membrane and a potential is also induced by iron restriction in 2001; PubMed Scopus Google Scholar). These genes are likely to specify a new system. cluster is also likely to represent a It of two genes a outer membrane and which is to be a that of the Fe2+-Fur repressed gene are with Fur of a gene that is at the to a gene that of the of I. Hantke K. J. PubMed Scopus Google Scholar). specific of is gene is with a well Fur box and its in S. is Fur repressed A.J. I. J. Bacteriol. 1995; 177: PubMed Google the Fe2+-Fur regulation also a at the with the gene of has an potential Fur box 2001; PubMed Scopus Google Scholar) with its Fe2+-Fur of a large number of genes encoding proteins involved in energy metabolism were found to be Fe2+-Fur-regulated genes and in only and have previously been to be N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar, I. Bäumler A.J. Hantke K. J. Mol. Biol. 1994; 236: 531-545Crossref PubMed Scopus (308) Google Scholar). C-P. Gunsalus R.P. Mol. Microbiol. 1995; PubMed Scopus Google Scholar) were induced by the Fe2+-Fur complex of which encode iron-containing respiratory complexes with a total of iron that Fur-dependent control of iron-containing respiratory proteins represents a iron mechanism whereby the production of a of iron proteins is regulated to iron availability. a mechanism the for iron to be under iron-restricted growth iron to be utilized and to that production of proteins not iron availability. with the data in has that the expression of and is by dip that for and an S. Gunsalus R.P. FEMS Microbiol. Lett. PubMed Google Scholar). However, in to the to be regulator was not S. Gunsalus R.P. FEMS Microbiol. Lett. PubMed Google Scholar). of the Fe2+-Fur induced genes in the and for S. Guest J.R. FEMS Rev. Scopus Google Scholar). the growth used were are low growth in J. Microbiol. PubMed Scopus Google Scholar) and thus It is also that were at the sample A potential is the of by the iron chelator and of the genes in S. Gunsalus R.P. FEMS Microbiol. Lett. PubMed Google Scholar). However, such an not be for the fur mutant and so not the dip and the fur expression effects in Fe2+-Fur induced genes in are not essential and the reductions in their expression growth not be to to a major growth mutants are to respiratory complexes, the to anaerobically and of expression by the Fe2+-Fur complex is likely to be indirect for most of the Fe2+-Fur induced genes and to be Fur It is that the Fur-dependent regulatory RNA as the regulator of these genes (11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar). It is that not genes to be induced by This the of some such as the for DNA It is also to that the expression of in the and the TCA is repressed by the Fe2+-Fur complex the TCA aconitase A that into is known to be induced by that E. coli respond to iron restriction by to iron of the gene is also Fur repressed in T. H. J. J.M. J. Microbiol. 2002; PubMed Scopus Google Scholar). Quantitative RT-PCR was used to the Fe2+-Fur expression of genes the of regulation were for the RT-PCR and array the of regulation and was generally for the RT-PCR the quantitative with with DNA showed Fe2+-Fur and thus the DNA array RT-PCR analysis of iron and Fur-dependent gene regulation Cellular Fe2+-Fur expression of is with the reduction in iron by the fur (5Abdul-Tehrani H. Hudson A.J. Chang Y-S. Timms A.R. Hawkins C. Williams J.M. Harrison P.M. Guest J.R. Andrews S.C. J. Bacteriol. 1999; 181: 1415-1428Crossref PubMed Google Scholar). of reduction in iron is due to low of FtnA iron and it was that the reduction in iron is due to expression of (5Abdul-Tehrani H. Hudson A.J. Chang Y-S. Timms A.R. Hawkins C. Williams J.M. Harrison P.M. Guest J.R. Andrews S.C. J. Bacteriol. 1999; 181: 1415-1428Crossref PubMed Google Scholar). This is by the array data in In to the fur to of a of the composition of wild-type and fur mutants grown in with was performed This analysis confirmed that the fur mutant have a as by analysis of the in of the wild-type the that Fur controls of as and (5Abdul-Tehrani H. Hudson A.J. Chang Y-S. Timms A.R. Hawkins C. Williams J.M. Harrison P.M. Guest J.R. Andrews S.C. J. Bacteriol. 1999; 181: 1415-1428Crossref PubMed Google Scholar, 11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar, S. Gunsalus R.P. FEMS Microbiol. Lett. PubMed Google Scholar). b and d and Fur and/or provide evidence for the Fe2+-Fur control of levels, spectroscopy was used to the of dip and/or the fur on the of and proteins and spectroscopy revealed that the cytochrome at nm is by to of by dip or the fur which well with the decrease in expression for the genes at nm is due to from I. J. PubMed Scopus Google cytochrome b of the cytochrome the cytochrome b of the cytochrome and b of succinate Neidhardt F.C. Escherichia coli and Salmonella: Cellular PubMed Scopus Google are at results from a of from the was at with to the other B, the at a the from the iron is results were for grown to OD650 nm 1.0 and with dip are not due to from a large from an electron paramagnetic spectroscopy of was used to the of proteins. the are as the proteins are found mainly in the In the major is the cluster that can be found for succinate and of the in the fur mutant is in the sample to be from succinate This well with the decrease in expression of the in the fur data showed of dip or the fur on expression and of Fe2+-Fur response is by RT-PCR on and However, array studies using to medium showed a expression of the as previously (11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google Scholar, C-P. Gunsalus R.P. Mol. Microbiol. 1995; PubMed Scopus Google Scholar). A at from iron in a is also in the samples most likely for is a iron Scopus Google Scholar). This was in the fur of is unknown it to a as previously in fur mutants K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). genes, from various other functional were also Fe2+-Fur only and have previously been to be Fe2+-Fur regulated N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar, J. Bacteriol. PubMed Scopus Google Scholar). is the Fe2+-Fur of the genes T. Microbiol. 2000; PubMed Scopus Google Scholar). known proteins in E. coli are which also iron, it is not that transport is induced by the Fe2+-Fur complex. the the array data only that of is iron and and data not However, is and how it is mediated is Interestingly, a gene cluster was Fe2+-Fur repressed a group of genes and within a gene cluster was induced by the Fe2+-Fur complex. This that gene be due to in intracellular iron or Fe2+-Fur encoding a iron-containing A. E. I. J. Mol. Microbiol. PubMed Scopus Google Scholar) appears to be Fe2+-Fur repressed which is by Fe2+-Fur repression of N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar). This that are three in E. have a role under iron-restricted conditions. Fe2+-Fur induced gene encoding an outer membrane of is also induced by iron in 2001; PubMed Scopus Google it is repressed by iron in S. T. H. J. J.M. J. Microbiol. 2002; PubMed Scopus Google Scholar). In to and are several other known Fe2+-Fur regulated genes flbB, fumC, purR, nohB, and that were not by dip and Fur in for are probably to the growth which are to expression of genes. expression is and and so and redox-stress for Guest J.R. Microbiol. 1994; PubMed Scopus Google and data that the purine is in that the Fe2+-Fur regulated of N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar) appears to be specific for (11Massé E. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4620-4625Crossref PubMed Scopus (854) Google which its of Fe2+-Fur control in has revealed that, despite is a to be iron metabolism in E. coli A major new role for Fur in iron has been in which Fur the control of iron-protein in response to iron availability. This new mechanism is in that it only involves gene It is how widespread mechanism its it is that it be with the capacity to gene expression in response to global transcriptional profiling studies on the effects of iron restriction and/or fur provide some evidence for iron regulation of genes in other In N. T. Mol. Microbiol. 2002; PubMed Scopus Google Scholar) several cytochrome systems and aconitase were to repressed by iron In S. a fur in expression of genes encoding proteins cytochrome B, cytochrome b involved in electron transport T. H. J. J.M. J. Microbiol. 2002; PubMed Scopus Google and in genes encoding proteins iron-sulfur protein, involved in energy metabolism and electron transport were iron restriction by 2001; PubMed Scopus Google Scholar). In genes were repressed by iron their have not been Mol. Microbiol. 2002; PubMed Scopus Google Scholar). K. Hantke for the of