Global Iron-dependent Gene Regulation in Escherichia coli
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m303381200
ISSN1083-351X
AutoresJonathan P. McHugh, Francisco Rodríguez‐Quiñones, Hossein Abdul-Tehrani, Dimitri A. Svistunenko, Robert K. Poole, Chris E. Cooper, Simon C. Andrews,
Tópico(s)Iron Metabolism and Disorders
ResumoOrganisms 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)ethyl]glycine; Fur, ferric uptake regulation; EPR, electron paramagnetic resonance.1The abbreviations used are: TCA, Tricarboxylic Acid Cycle; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 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 & Molecular Biology. 2nd Ed. ASM Press, Washington, D. C.1996: 1075-1090Google Scholar). Ferri-siderophore complexes are taken up via specific outer membrane receptors in a process that is driven by the inner membrane potential and mediated by the energy-transducing TonB-ExbB-ExbD system. Periplasmic-binding proteins shuttle ferri-siderophores from the receptors to inner membrane ABC transporters that, in turn, deliver the ferri-siderophores to the cytosol where the complexes are probably dissociated by reduction. E. coli has six known siderophore receptors (Cir, FecA, FepA, FhuA, FhuE, Fiu) providing specificity for several ferri-siderophores (and ferric dicitrate) of which only enterobactin and its derivatives are synthesized endogenously (4Hantke K. Curr. Opin. Microbiol. 2001; 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 template or reverse-transcriptase free controls. The template RNA samples were prepared separately from those used for macroarray analysis. Macroarray Hybridization and Scanning—Labeled cDNA was hybridized independently to two Panorama™ E. coli gene arrays (Sigma-Genosys) essentially as described by the manufacturer. Hybridized and washed arrays were then exposed to low intensity phosphorimaging cassettes (Molecular Dynamics) for 45 h and the cassettes were then scanned with a Phosphoimager SI system (Molecular Dynamics) at 200-μm resolution. Filters were stripped prior to re-use as described by the manufacturer. Data Analysis—The phosphoimager image files were analyzed using ImageQuant software (Molecular Dynamics). Pixel intensities for each spot were manipulated using Microsoft Excel. The 632 blank spots were used to calculate the background, which was then subtracted from the 8580 gene-specific spots (each gene present in duplicate). Spots with intensities less than two standard deviations above mean background values were considered to display no significant expression. The intensity of each of the gene-specific spots within an individual array was normalized by expressing values as percentages of total gene-specific spot intensity. This allowed comparisons between array experiments. Array experiments were performed in duplicate, and average values calculated along with confidence levels (Student's t test values). Whole Cell Spectroscopy—E. coli wild type (MC4100) and fur mutant (MC4100 fur) were grown aerobically in L broth, with or without 200 μm dip, to mid-log (OD650, 0.5), late-log (OD650, 1.0), and stationary phase (OD650, 3.0). Cells were harvested by centrifugation and 0.5 g of wet cells were resuspended in 10 ml of 0.1 m phosphate buffer (pH 7). Samples of 4 ml were oxidized with 0.005% (w/v) potassium ferricyanide and 0.005% (w/v) ammonium persulfate or were reduced with 0.005% (w/v) sodium dithionite. Spectra were then recorded at room temperature using a dual beam spectrophotometer over a wavelength range of 400–700 nm, with a reference wavelength of 500 nm (13Kalnenieks U. Galinina N. Bringer-Meyer S. Poole R.K. FEMS Microbiol. Lett. 1998; 168: 91-97PubMed Google Scholar). Cytochrome d- and b-type cytochrome levels were quantified as previously described (14Jones C.W. Redfearn E.R. Biochim. Biophys. Acta. 1966; 113: 467-481Crossref PubMed Google Scholar, 15Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3369-3374Google Scholar). Prior to whole cell EPR spectroscopy, cells were washed in 0.1 mm phosphate buffer (pH 7) containing 50 mm EDTA in order to remove Mn from the cell surface. Half of each sample was reduced by the addition of 0.005% (w/v) sodium dithionite followed by repetitive freeze-thawing. The EPR spectra were measured at 10 K on a Bruker EMX EPR spectrometer equipped with an Oxford Instruments liquid helium system. A spherical high quality Bruker resonator SP9703 was used. The EPR samples were frozen in Wilmad SQ EPR tubes. Measurements were as follows: microwave power, 3.18 milliwatts; microwave frequency, 9.46 GHz; modulation frequency, 100 kHz; modulation amplitude, 3 G; sweep rate, 3.58 G/s; time constant, 0.082 s. Protein concentrations were determined as previously described (16Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5279) Google Scholar). Native Gel Electrophoresis of 55Fe-labeled Soluble E. coli Proteins—E. coli cultures were grown in 5 ml of L broth containing 55FeC13 (∼0.5 MBq/ml). Cells (5 OD650 nm units) were harvested by centrifugation in the postexponential phase and washed in saline at 4 °C. Soluble cell extracts were then prepared by the spheroplast osmotic lysis method (17Philips-Jones M.K. Watson F.J. Martin R. J. Mol. Biol. 1993; 233: 1-6Crossref PubMed Scopus (28) Google Scholar) with the following modifications. Tricine was used in place of Tris, dithiothreitol was omitted, 0.1% Triton X-100 was included at the lysis stage and glycerol was added postlysis to a final concentration of 5%. Soluble cell extracts were electrophoresed in native-acrylamide gels containing 0.1% Triton X-100 and Tricine in place of Tris. Gels were then dried under vacuum and autoradiographed with Kodak Biomax MS film. Identification of Fe2 + -Fur-regulated Genes by Transcriptional Profiling—Genomic transcriptional profiling was used to identify genes regulated by both Fe and Fur (see "Materials and Methods"). The transcription profile of MC4100 (wild-type) grown in rich broth was compared with those of both the wild-type grown with an Fe2+ chelator (dip) and the fur mutant (MC4100 fur) grown without chelator. Samples were harvested at an OD650 nm of 1.0, corresponding to early postexponential phase. No major growth differences were observed for the three experimental conditions. 33P-labeled cDNA was prepared from the RNA samples using reverse transcriptase and random hexaoligonucleotides, and was hybridized to E. coli Panorama Macroarrays. Each array experiment was performed in duplicate using pooled RNA samples prepared from three identical cultures. Duplicate experiments gave good reproducibility with correlation coefficients of 0.97. Comparison of the L broth versus L-broth plus dip, or wild-type versus fur mutant, gave lower correlation coefficients (0.95 and 0.90). Since members of the Fur modulon are regulated by iron and Fur in conjunction, only those genes that were ≥2-fold regulated by both dip and the fur mutation are considered further here. Accordingly, 101 genes were found to be regulated by the Fe2+-Fur complex of which 53 were repressed and 48 induced. These genes fall into three major categories: iron metabolism (Table I), energy production (Table II), and miscellaneous/unknown (Table IV).Table IIron and Fur regulation of genes involved in iron metabolismTable IIIron- and Fur-regulated genes involved in energy metabolismTable IIIron- and Fur-regulated genes involved in energy metabolismTable IVMiscellaneous iron- and Fur-regulated genesTable IVMiscellaneous iron- and Fur-regulated genes Iron Metabolism: Potential Novel Iron Transporters—Reassuringly, most of the known iron-acquisition genes were induced by both the chelator and the fur mutation (Table I) validating the experimental procedure. The enterobactin biosynthesis (entA–F) genes were among the most highly de-repressed (average ∼21-fold) genes (Table I), presumably to ensure that energy is not needlessly squandered on enterobactin production during conditions of iron sufficiency. In contrast, the ferric-enterobactin uptake genes (fepABCDEG) were weakly de-repressed (average ∼2-fold) indicating that enterobactin production systems are more strongly controlled by iron than the ferri-enterobactin acquisition apparatus. As previously observed, the fecIRABCDE, feoAB, fhuE, fhuF, and cirA genes involved in ferric-dicitrate, ferrous iron, ferri-coprogen/rhodotorulic acid, ferrioxamine B, and ferric-dihydroxybenzoate utilization were all repressed by the Fe2+-Fur complex, as were the tonB and exbBD genes required for energy-dependent ferrisiderophore transfer across the outer membrane. However, the fhuACDB operon, specifying the ferric hydroxamate uptake apparatus, was not significantly affected by the chelator or fur mutation although it is known that fhuA-lacZ transcriptional fusions are Fe2+-Fur regulated (19Hantke K. Mol. Gen. Genet. 1981; 182: 288-292Crossref PubMed Scopus (368) Google Scholar). The reason for this discrepancy is unclear, but could be related to growth-phase effects. The suf operon, which probably functions in iron-sulfur cluster assembly during iron starvation and redox stress (20Zheng M. Wang X. Templeton L.J. Smulski D.R. LaRossa R.A. Storz G. J. Bacteriol. 2001; 183: 4562-4570Crossref PubMed Scopus (647) Google Scholar), was also Fe2+-Fur repressed (average of 5-fold), as previously indicated (21Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar). However, the iscSUA-hscBA-fdx cluster, which encodes genes with a housekeeping role in iron-sulfur cluster assembly, was not Fe2+-Fur controlled (not shown). Appropriately, the bfd gene encoding a ferredoxin thought to be involved in iron release from bacterioferritin, was de-repressed (22Quail M.A. Jordan P. Grogan J.M. Butt J.N. Lutz M. Thomson A.J. Andrews S.C. Guest J.R. Biochem. Biophys. Res. Commun. 1996; 229: 635-642Crossref PubMed Scopus (46) Google Scholar) whereas the ftnA gene specifying the iron-storage protein, ferritin A, was repressed by the chelator and fur mutation, as previously observed (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 significant absentee from the list of genes in Table I is bfr, coding for bacterioferritin. Although this gene is known to be Fe2+-Fur induced, its expression is RpoS dependent and so is restricted to the stationary phase. 2J. Grogan and S. C. Andrews, unpublished observations. Thus, the array data are generally consistent with numerous previous expression studies on the Fur-regulated components of iron metabolism. The array analysis enabled the identification of several unknown genes with potential functions in iron acquisition (Table I). These were initially recognized either because of their chelator- and fur-dependent expression or by their chromosomal co-location with such genes. They are organized into 6 clusters (boxed in Table I), of which the largest (ybiM-ybiLXI-ybiJ) consists of 5 co-polar genes encoding: YbiM and YbiJ, two putative-periplasmic/exported proteins that are related to each other, but not to any other E. coli protein; YbiL (or Fiu), a probable TonB-dependent outer membrane receptor previously shown to be involved in Fe3+-dihydroxybenzoylserine and -dihydroxybenzoate utilization (23Hantke K. FEMS Microbiol. Lett. 1990; 67: 5-8Google Scholar); YbiX, a homologue of a protein encoded by an Fe-repressed Pseudomomas aeruginosa gene (piuC) thought to be involved in iron uptake (24Ochsner U.A. Vasil M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4409-4414Crossref PubMed Scopus (210) Google Scholar); and YbiI, a probable C4-Zn finger protein of unknown function. The above suggests that the Fiu-mediated Fe-uptake system is more complex than hitherto believed. The second cluster (ycdN-ycdOB) consists of three copolar genes encoding: YcdN, a homologue (24% amino acid sequence identity) of the high-affinity ferrous iron transporter (Ftr1p) of yeast; YcdO, a potential exported lipoprotein of unknown function; and YcdB, another potentially exported protein of unknown function. A twin arginine transporter (tat) motif was previously identified in the N terminus of YcbB (25Stanley N.R. Findlay K. Berks B.C. Palmer T. J. Bacteriol. 2001; 183: 139-144Crossref PubMed Scopus (140) Google Scholar), and a similar motif is present in the N terminus of YcdO. This suggests, as for many other tat-exported proteins, that these proteins could possess prosthetic groups inserted prior to export to the periplasm. Homologues of the ycdNOB genes are found co-located in the chromosomes of at least 7 other bacteria indicating that these 3 genes form a functional unit. YcdB bears homology to STY2683 of Salmonella typhimurium, a putative iron-dependent peroxidase. By analogy with the yeast Ftr1p system (26Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (570) Google Scholar), we speculate that YcdN acts as a ferrous iron transporter, and that YcdO and YcdB act together as a novel periplasmic iron oxidase or reductase (this possibility is currently being tested). The third cluster consists of three genes, yddAB-pqqL, that appear to form an operon encoding: YddA, which is homologous to ABC transporters; YddB, homologous to TonB-dependent outer membrane receptors; and PqqL, a potential Zn peptidase (pqqL is also induced by iron restriction in Pasteurella multocia; 45Paustian M.L. May B.J. Kapur V. Infect. Immun. 2001; 69: 4109-4115Crossref PubMed Scopus (77) Google Scholar). These genes are likely to specify a new 3-component iron-uptake system. The fourth cluster is also likely to represent a newly identified iron-uptake pathway. It consists of two divergently arranged genes encoding: YcnD, a probable TonB-dependent outer membrane receptor; and YncE, which is predicted to be a pyrolo-quinoline quinone containing periplasmic oxidase. Note that all four of the above Fe2+-Fur repressed gene clusters are associated with predicted Fur boxes. The fifth locus consists of a single gene (ydiE) that is related (36% identity at the amino acid sequence level) to hemP, a gene that forms part of the heme utilization operon (hemPRST) of Yersinia enterocolitica (27Stojiljkovic I. Hantke K. EMBO J. 1992; 11: 4359-4367Crossref PubMed Scopus (200) Google Scholar). The specific function of hemP is uncertain. The ydiE gene is associated with a well predicted Fur box and its homologue (hemP) in S. typhimurium is Fur repressed (28Tsolis R.M. Baumler A.J. Stojiljkovic I. Heffron F. J. Bacteriol. 1995; 177: 4628-4637Crossref PubMed Google Scholar), supporting the Fe2+-Fur dependent regulation observed here. The final locus also comprises a single gene, yqjH, homologous (28% identity at the amino acid sequence level) with the siderophore-utilization gene (viuB) of Vibrio cholerae. yqjH has an appropriately positioned potential Fur box (29Panina E.M. Mironov A.A. Gelfand M.S. Nucleic Acids Res. 2001; 29: 5195-5206Crossref PubMed Scopus (81) Google Scholar) consistent with its Fe2+-Fur dependence. Energy Metabolism: Control of Iron-rich Proteins—Unexpectedly, a large number of genes encoding proteins involved in energy metabolism were found to be Fe2+-Fur-regulated (Table II). Of those genes and operons listed in Table II, only cyoA and gpmA have previously been reported to be Fe2+-Fur-controlled (8Vassinova N. Kozyruv D. Microbiol. 2000; 146: 3171-3182Crossref PubMed Scopus (73) Google Scholar, 6Stojiljkovic I. Bäumler A.J. Hantke K. J. Mol. Biol. 1994; 236: 531-545Crossref PubMed Scopus (308) Google Scholar). Most (36Park S-J Tseng C-P. Gunsalus R.P. Mol. Microbiol. 1995; 15: 473-482Crossref PubMed Scopus (110) Google Scholar) were induced by the Fe2+-Fur complex of which 32 encode iron-containing respiratory complexes associated with a total of 148 iron atoms (per subunit). We speculate that this Fur-dependent control of iron-containing respiratory proteins represents a newly recognized iron homeostatic mechanism whereby the production of a subset of iron proteins is regulated according to iron availability. Such a mechanism would allow the cellular demand for iron to be reduced under iron-restricted growth conditions, enabling available iron to be utilized more economically and helping to ensure that production of Fe-requiring proteins does not exceed iron availability. Partly consistent with the data in Table II, previous work has shown that the anaerobic expression of cydA, cyoA, narG, and frdA is 2–14-fold reduced by dip (note that for cyoA and cydA this effect required an fnr background) (30Cotter P.A. Darie S. Gunsalus R.P. FEMS Microbiol. Lett. 1992; 100: 227-232Crossref PubMed Google Scholar). However, in contrast to the findings presented here, this effect appeared to be Fur-independent (the iron-dependent regulator was not identified; Ref. 30Cotter P.A. Darie S. Gunsalus R.P. FEMS Microbiol. Lett. 1992; 100: 227-232Crossref PubMed Google Scholar). Nearly all of the Fe2+-Fur induced genes in Table II require the anaerobic regulator, Fnr, and anaerobiosis for full induction (31Spiro S. Guest J.R. FEMS Microbiol Rev. 1990; 75: 399-428Crossref Scopus (189) Google Scholar). Although the growth conditions used here were aerobic, O2 tensions are very low during late-log growth in rich broth (32Rainnie D.J. Bragg P.D. J. Gen. Microbiol. 1973; 77: 339-349Crossref PubMed Scopus (23) Google Scholar) and thus could favor Fnr-dependent expression. It is also possible that anaerobic conditions were introduced at the sample harvesting stage. A potential complication is the possible inactivation of Fnr by the iron chelator and consequent down-regulation of the Fnr-dependent genes in Table II (30Cotter P.A. Darie S. Gunsalus R.P. FEMS Microbiol. Lett. 1992; 100: 227-232Crossref PubMed Google Scholar). However, such an effect would not be anticipated for the fur mutant and so would not explain both the dip and the fur expression effects shown in Table II. The Fe2+-Fur induced genes listed in Table II are not essential and the observed reductions in their expression during aerobic growth would not be expected to lead to a major growth defect. Indeed, although fnr mutants are unable to express anaerobic respiratory complexes, they retain the ability to grow both anaerobically (via fermentation) and aerobically. The induction of expression by the Fe2+-Fur complex is likely to be indirect since for most of the Fe2+-Fur induced genes (Tables I, II, and IV), there appear to be no associated Fur boxes. It is probable that the recently identified Fur-dependent regulatory RNA molecule, RyhB, acts as the direct 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 noteworthy that not all Fe-protein-encoding genes appear to be induced by Fe2+-Fur. This may reflect the high importance of some Fe-proteins, such as the aerobic ribonucleotide reductase (NrdAB) required for DNA biosysnthesis. It is also interesting to note that the expression of gpmA (encoding phosphoglycerate mutase in the glycolytic pathway) and gltA (encoding the TCA cycle enzyme, citrate synthase) is repressed by the Fe2+-Fur complex (Table II), whereas acnA (encoding the TCA cycle enzyme aconitase A that converts citrate into isocitrate) is known to be induced by Fe2+-Fur, suggesting that E. coli may respond to iron restriction by producing citrate to mediate iron uptake. Expression of the citrate synthase gene (prpC) is also Fur repressed in Shewanella oneidensis (44Thompson D.K. Beliaev A.S. Giometti C.S. Tollaksen S.L. Khare T. Lies D.P. Nealson K.H. Lim H. Yates III, J. Brandt C.C. Tiedje J.M. Zhou J. Appl. Environ. Microbiol. 2002; 68: 881-892Crossref PubMed Scopus (146) Google Scholar). Quantitative RT-PCR was used to confirm the Fe2+-Fur dependent expression of 12 iron-regulated genes (Table III). Although the directions of regulation were identical for the RT-PCR and array data, the degree of regulation varied considerably and was generally greater for the RT-PCR analysis, presumably reflecting the higher quantitative precision obtained with RT-PCR. Control experiments with polA (encoding DNA polymerase I) showed no significant Fe2+-Fur e
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