The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR
2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês
10.1074/jbc.m508693200
ISSN1083-351X
AutoresJulia Wennerhold, Andreas Krug, Michael Bott,
Tópico(s)Enzyme Structure and Function
ResumoThe mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum ΔripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the ΔripA mutant and the wild type revealed that the acn mRNA level was increased in the ΔripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the ΔripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). From 13 identified binding sites, the RipA consensus binding motif RRGCGN4RYGAC was deduced. Expression of ripA itself is repressed under iron excess by DtxR, since purified DtxR binds to a well conserved binding site upstream of ripA. Thus, repression of acn and the other target genes indicated above under iron limitation involves a regulatory cascade of two repressors, DtxR and its target RipA. The modulation of the intracellular iron usage by RipA supplements mechanisms for iron acquisition that are directly regulated by DtxR. The mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum ΔripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the ΔripA mutant and the wild type revealed that the acn mRNA level was increased in the ΔripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the ΔripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). From 13 identified binding sites, the RipA consensus binding motif RRGCGN4RYGAC was deduced. Expression of ripA itself is repressed under iron excess by DtxR, since purified DtxR binds to a well conserved binding site upstream of ripA. Thus, repression of acn and the other target genes indicated above under iron limitation involves a regulatory cascade of two repressors, DtxR and its target RipA. The modulation of the intracellular iron usage by RipA supplements mechanisms for iron acquisition that are directly regulated by DtxR. Corynebacterium glutamicum is a nonpathogenic, aerobic Gram-positive soil bacterium that is used for large scale industrial production of amino acids, predominantly l-glutamate (1.5 million tons/year) and l-lysine (0.7 million tons/year). In addition, C. glutamicum has gained interest as a suitable model organism for the Corynebacterineae, a suborder of the actinomycetes that includes the genus Mycobacterium. An overview on C. glutamicum biology, genetics, physiology, and biotechnology can be found in a recent monograph (1Eggeling L. Bott M. Handbook of Corynebacterium glutamicum. CRC Press, Taylor & Francis Group, Boca Raton, FL2005Crossref Google Scholar). The citric acid cycle is of central importance for metabolism in general and for amino acid production in particular, because it provides the biosynthetic precursors of the aspartate and glutamate family of amino acids. Despite its key role, knowledge about the genetic regulation of this pathway in C. glutamicum is scarce. We recently could show that the activity of aconitase (EC 4.2.1.3), which catalyzes the stereospecific and reversible isomerization of citrate to isocitrate via cis-aconitate, varies depending on the carbon source and that this is caused by transcriptional regulation (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). A repressor of the TetR family, called AcnR, was identified, which represses aconitase by binding to an imperfect inverted repeat within the acn promoter region and interfering with the binding of RNA polymerase (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The factors that control binding of AcnR to its operator are not yet known. DNA microarray experiments revealed that acn expression is not only influenced by the carbon source but also by the iron concentration of the medium (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Under iron limitation, the acn mRNA level in the wild type was 3-fold lower than under iron excess. In the ΔacnR mutant, this decrease was even larger (4.8-fold), presumably because the increased expression of aconitase, which contains a 4Fe-4S cluster, leads to an enhanced iron starvation. We now have identified a new transcriptional regulator, designated RipA, which is responsible for iron-dependent regulation of aconitase and several other iron-containing proteins. Evidence is provided that RipA represses acn and six other target operons under iron limitation and is itself repressed under iron excess by the global iron repressor DtxR. Bacterial Strains, Media, and Growth Conditions—All strains and plasmids used in this work are listed in supplemental Table S1. The C. glutamicum type strain ATCC13032 (3Kinoshita S. Udaka S. Shimono M. J. Gen. Appl. Microbiol. 1957; 3: 193-205Crossref Scopus (411) Google Scholar) was used as wild type. Strain ΔripA is a derivative containing an in-frame deletion of the ripA gene. For growth experiments, 5 ml of brain heart infusion medium (Difco) was inoculated with colonies from a fresh LB agar plate (4Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and incubated for 6 h at 30°C. After washing, the cells of this first preculture were used to inoculate a 500-ml shake flask containing 50 ml of CGXII minimal medium (5Keilhauer C. Eggeling L. Sahm H. J. Bacteriol. 1993; 175: 5595-5603Crossref PubMed Google Scholar) with 4% (w/v) glucose and either 1 μm FeSO4 (iron starvation) or 100 μm FeSO4 (iron excess). This second preculture was cultivated overnight at 30 °C and then used to inoculate the main culture to an A600 ∼1. The main culture contained the same iron concentration as the second preculture. The trace element solution with iron salts omitted and the FeSO4 solution were always added after autoclaving. For growth of C. glutamicum strains carrying plasmid pJC1 or pJC1-ripA, the medium was supplemented with 25 μg/ml kanamycin. For all cloning purposes, Escherichia coli DH5 (Invitrogen) was used as host, for overproduction of RipA and DtxR E. coli BL21(DE3) (6Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4772) Google Scholar). The E. coli strains were cultivated aerobically in LB medium at 37 °C (DH5) or at 30 °C (BL21(DE3)). When appropriate, kanamycin was added to a concentration of 50 μg/ml. Recombinant DNA Work—The enzymes for recombinant DNA work were obtained from Roche Applied Science or New England Biolabs (Frankfurt, Germany). The oligonucleotides used in this study were obtained from Operon (Cologne, Germany) and are listed in supplemental Table S2. Routine methods like PCR, restriction, or ligation were carried out according to standard protocols (4Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Chromosomal DNA from C. glutamicum was prepared as described (7Eikmanns B.J. Thum-Schmitz N. Eggeling L. Luedtke K.U. Sahm H. Microbiology. 1994; 140: 1817-1828Crossref PubMed Scopus (231) Google Scholar). Plasmids from E. coli were isolated with the QIAprep spin miniprep kit (Qiagen, Hilden, Germany). E. coli was transformed by the RbCl method (8Hanahan D. DNA Cloning. 1. IRL Press, Oxford, UK1985: 109-135Google Scholar), and C. glutamicum was transformed by electroporation (9van der Rest M.E. Lange C. Molenaar D. Appl. Microbiol. Biotechnol. 1999; 52: 541-545Crossref PubMed Scopus (359) Google Scholar). DNA sequencing was performed with a Genetic Analyzer 3100-Avant (Applied Biosystems, Darmstadt, Germany). Sequencing reactions were carried out with the Thermo Sequenase primer cycle sequencing kit (Amersham Biosciences). An in-frame ripA deletion mutant of C. glutamicum was constructed via a two-step homologous recombination procedure as described previously (10Niebisch A. Bott M. Arch. Microbiol. 2001; 175: 282-294Crossref PubMed Scopus (150) Google Scholar). The ripA up- and downstream regions (∼500 bp each) were amplified using the oligonucleotide pairs orf1558-A-for/orf1558-B-rev and orf1558-C-for/orf1558-D-rev, respectively, and the products served as template for cross-over PCR with oligonucleotides orf1558-A-for and orf1558-D-rev. The resulting PCR product of ∼1 kb was digested with EcoRI and HindIII and cloned into pK19mobsacB (11Schäfer A. Tauch A. Jäger W. Kalinowski J. Thierbach G. Pühler A. Gene (Amst.). 1994; 145: 69-73Crossref PubMed Scopus (2098) Google Scholar). DNA sequence analysis confirmed that the cloned PCR product did not contain spurious mutations. Transfer of the resulting plasmid pK19mobsacB-ΔripA into C. glutamicum and screening for the first and second recombination event were performed as described previously (10Niebisch A. Bott M. Arch. Microbiol. 2001; 175: 282-294Crossref PubMed Scopus (150) Google Scholar). Kanamycin-sensitive and saccharose-resistant clones were tested by PCR analysis of chromosomal DNA with the primer pair orf1558-amp-for/orf1558-amp-rev (supplemental Table S2). Of 10 clones tested, five showed the wild-type situation (2.0-kb fragment) and five had the desired in-frame deletion of the ripA gene (1.1-kb fragment), in which all nucleotides except for the first six codons and the last 12 codons were replaced by a 21-bp tag. In order to complement the ΔripA mutant, the ripA coding region and 250-bp upstream DNA containing the promoter region were amplified using oligonucleotides (ripA+250-for (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and ripA+250-rev) introducing a SalI and a PstI restriction site, respectively. The resulting 1245-bp PCR product was cloned into the vector pJC1 (12Cremer J. Eggeling L. Sahm H. Mol. Gen. Genet. 1990; 220: 478-480Crossref Scopus (71) Google Scholar). The resulting plasmid pJC1-ripA and pJC1 were used to transform C. glutamicum wild type and the ΔripA strain. For overproduction and purification of RipA with an N-terminal StrepTag-II (13Skerra A. Schmidt T.G. Methods Enzymol. 2000; 326: 271-304Crossref PubMed Google Scholar), the ripA coding region was amplified using oligonucleotides that introduce an NdeI restriction site, including the start codon (ripA-2-for) and an XhoI restriction site after the stop codon (ripA-2-rev). The purified PCR product was cloned in the modified expression vector pET28b-Streptag (14Engels S. Schweitzer J.E. Ludwig C. Bott M. Schaffer S. Mol. Microbiol. 2004; 52: 285-302Crossref PubMed Scopus (116) Google Scholar), resulting in plasmid pET28b-Streptag-ripA. The RipA protein encoded by this plasmid contains 14 additional amino acids (MASWSHPQFEKGAH) at the amino terminus. For overproduction and purification of DtxR, the dtxR coding region (equivalent to NCgl1845) was amplified using oligonucleotides that introduced an NdeI restriction site at the translation initiation codon (dtxR-for-1) and four histidine codons plus an XhoI restriction site before the stop codon (dtxR-rev-1). The PCR product was cloned into the pET24b vector, resulting in plasmid pET24b-dtxR-C. The DtxR protein encoded by this plasmid contains 12 additional amino acids at the carboxyl terminus (HHHHLEHHHHHH). The PCR-derived portions of pET28b-Streptag-ripA and pET24b-dtxR-C were analyzed by DNA sequence analysis and found to contain no spurious mutations. For overproduction of RipA and DtxR, the two plasmids were transferred into E. coli BL21(DE3). Preparation of Total RNA—Cultures of the wild type and the ΔripA mutant were grown in CGXII minimal medium containing 4% (w/v) glucose under iron limitation (1 μm FeSO4) or iron excess (100 μm FeSO4). In the exponential growth phase at an A600 of 4–6, 25 ml of the cultures were used for the preparation of total RNA as described previously (15Möker N. Brocker M. Schaffer S. Krämer R. Morbach S. Bott M. Mol. Microbiol. 2004; 54: 420-438Crossref PubMed Scopus (131) Google Scholar). Isolated RNA samples were analyzed for quantity and quality by UV spectrophotometry and denaturing formaldehyde-agarose gel electrophoresis (4Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), respectively, and stored at -70 °C until use. DNA Microarray Analyses—The generation of whole-genome DNA microarrays (16Wendisch V.F. J. Biotechnol. 2003; 104: 273-285Crossref PubMed Scopus (114) Google Scholar), synthesis of fluorescently labeled cDNA from total RNA, microarray hybridization, washing, and data analysis were performed as described previously (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Lange C. Rittmann D. Wendisch V.F. Bott M. Sahm H. Appl. Environ. Microbiol. 2003; 69: 2521-2532Crossref PubMed Scopus (72) Google Scholar, 18Polen T. Wendisch V.F. Appl. Biochem. Biotech. 2004; 118: 215-232Crossref PubMed Scopus (53) Google Scholar, 19Ishige T. Krause M. Bott M. Wendisch V.F. Sahm H. J. Bacteriol. 2003; 185: 4519-4529Crossref PubMed Scopus (125) Google Scholar). Genes that exhibited significantly changed mRNA levels (p < 0.05 in a Student's t test) by at least a factor of 1.7 were determined in two series of DNA microarray experiments: (i) five comparisons of the wild type and the ΔripA mutant cultivated in CGXII minimal medium with 4% (w/v) glucose under iron limitation (1 mm FeSO4); (ii) two comparisons of the wild type and the ΔripA mutant cultivated in CGXII-glucose medium under iron excess (100 μm FeSO4). Aconitase Assay—Aconitase activity was determined as the rate of cis-aconitate formation from isocitrate (20Henson C.P. Cleland W.W. J. Biol. Chem. 1967; 242: 3833Abstract Full Text PDF PubMed Google Scholar), as described previously (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), except that the assay was performed at 30 °C. Cells of the 20-ml main culture were harvested by centrifugation at 5,000 × g for 10 min and 4 °C. The cell pellet was resuspended in 90 mm Tris/HCl, pH 8.0, and used for the preparation of cell extract. The assay mixture contained 950–995 μl of 90 mm Tris/HCl, pH 8.0, and 20 mm dl-trisodium isocitrate. The reaction was started with the addition of 5–50 μl of cell extract, and the formation of cis-aconitate was followed by measuring the absorbance increase at 240 nm using a Jasco V560 spectrophotometer. An extinction coefficient for cis-aconitate of 3.6 mm-1 cm-1 at 240 nm was used. One unit of activity corresponds to 1 μmol of isocitrate converted to cis-aconitate per min. Overproduction and Purification of RipA—E. coli BL21(DE3) carrying the plasmid pET28b-strep-ripA was grown at 30 °C in 200 ml of LB medium with 50 μg/ml kanamycin to an A600 of ∼1.2 before adding 1 mm isopropyl β-d-thiogalactoside. After cultivation for another 4 h, cells were harvested by centrifugation, washed once, and stored at -20 °C. For cell extract preparation, thawed cells were resuspended in 10 ml of buffer W (100 mm Tris/HCl, pH 8.0, 150 mm NaCl). After the addition of 1 mm diisopropylfluorophosphate and 1 mm phenylmethylsulfonyl fluoride, the cell suspension was passed three times through a French pressure cell (SLM Aminco, Spectronic Instruments, Rochester, NY) at 207 megapascals. Intact cells and cell debris were removed by centrifugation (15 min, 5,000 × g, 4 °C), and the cell-free extract was subjected to ultracentrifugation (1 h, 150,000 × g, 4 °C). The supernatant obtained after ultracentrifugation was applied to a StrepTactin-Sepharose column with a bed volume of 1 ml (IBA, Göttingen, Germany). The column was washed with 6 ml of buffer W, and RipA tagged with StrepTag-II was eluted with 8 × 0.5 ml of buffer W containing 7.5 mm desthiobiotin (Sigma). Fractions containing RipA were pooled, and the buffer was exchanged against TG buffer (30 mm Tris/HCl, pH 7.5, 10% (v/v) glycerin) using Vivaspin concentrators with a cut-off of 10 kDa. Protein concentrations were determined with the BCA protein assay kit (Pierce) using bovine serum albumin as a standard. The purity of the protein preparations was assessed by SDS-PAGE and subsequent protein detection with Gel Code blue stain reagent (Pierce). Using this protocol, ∼0.2 mg of RipA protein was purified to apparent homogeneity from 200 ml of culture. Overproduction and Purification of DtxR—E. coli BL21(DE3) carrying the plasmid pET24b-dtxR was grown at 30 °C in 100 ml of LB with 50 μg/ml kanamycin. Expression was induced at an A600 of ∼0.3 with 1 mm isopropyl β-d-thiogalactoside. Four hours after induction, cells were harvested by centrifugation and stored at -20 °C. For cell extract preparation, thawed cells were washed once and resuspended in 10 ml of TNGI5 buffer (20 mm Tris/HCl, pH 7.9, 300 mm NaCl, 5% (v/v) glycerol, 5 mm imidazol) containing 1 mm diisopropylfluorophosphate and 1 mm phenylmethylsulfonyl fluoride. Disruption of the cells and fractionation by centrifugation was performed as described above for RipA purification. DtxR present in the supernatant of the ultracentrifugation step was purified by nickel affinity chromatography using nickel-activated nitrilotriacetic acid-agarose (Novagen). After washing the column with TNGI50 buffer (which contains 50 mm imidazol), DtxR protein was eluted with TNGI100 buffer (which contains 100 mm imidazol). Fractions containing DtxR were pooled, and the elution buffer was exchanged against TG buffer (30 mm Tris/HCl, pH 7.5, 10% (v/v) glycerin). From 100 ml of culture, ∼3 mg of DtxR was purified to apparent homogeneity. Gel Shift Assays—For band shift assays of RipA with putative target promoters, purified RipA protein was mixed with DNA fragments (200–630 bp, final concentration 8–13 nm) in a total volume of 20 μl. The binding buffer contained 20 mm Tris/HCl, pH 7.5, 0.5 mm EDTA, 5% (v/v) glycerol, 1 mm dithiothreitol, 0.005% (v/v) Triton X-100, 50 mm NaCl, 5 mm MgCl2, and 2.5 mm CaCl2. Approximately 20 nm of different nontarget promoter fragments (clpC, clpP, ripA, and porB) were added as a negative control. After incubation for 30 min at room temperature, the samples were separated on a 10% native polyacrylamide gel at room temperature and 170 V using 1× TBE (89 mm Tris base, 89 mm boric acid, 2 mm EDTA) as electrophoresis buffer. The gels were subsequently stained with Sybr Green I according to the instructions of the supplier (Sigma) and photographed. Binding of DtxR to the ripA promoter was carried out in a 20-μl reaction mixture containing 100 mm Tris/HCl (pH 7.5), 5 mm MgCl2,40 mm KCl, 10% (v/v) glycerol, 1 mm dithiothreitol, 150 μm MnCl2, an 18 nm concentration of a 300-bp ripA promoter DNA fragment, and DtxR in concentrations ranging from 0 to 3.6 μm. The ripA fragment covered the region from position -230 to +70 relative to the translation start and was obtained by PCR with primers ripA-Prom-for and ripA-Prom-rev. As a negative control, a 23 nm concentration of a 200-bp acn promoter fragment extending from position +190 to -50 relative to the acn transcription start site (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) was added. This fragment was amplified with primers acn-Prom4-for and acn-Prom4-rev. The reaction mixture was incubated at room temperature for 30 min and then loaded onto a 10% native polyacrylamide gel containing 1 mm dithiothreitol and 150 μm MnCl2. Electrophoresis was performed at room temperature and 170 V using 1× TB (89 mm Tris base, 89 mm boric acid) supplemented with 1 mm dithiothreitol and 150 μm MnCl2 as electrophoresis buffer. All PCR products used in the gel shift assays were purified with the PCR purification Kit (Qiagen, Hilden, Germany) and eluted in EB buffer (10 mm Tris/HCl, pH 8.5). Identification of RipA as a Potential Iron-dependent Regulator of the Aconitase Gene—In a previous study, we showed that expression of the aconitase gene acn of C. glutamicum is influenced by the iron availability, being reduced under iron limitation (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This regulation also occurred in a mutant lacking AcnR, a repressor of the acn gene, and thus must be mediated by a different regulator or regulatory mechanism. A candidate gene that might be responsible for iron-dependent regulation of acn was identified in the DNA microarray experiments used to compare the gene expression profile of C. glutamicum under iron excess and iron limitation. Expression of the gene NCgl0943 was strongly influenced by the iron availability (2Krug A. Wendisch V.F. Bott M. J. Biol. Chem. 2005; 280: 585-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Its mRNA level was always found to be increased under iron-limiting conditions, and it thus behaved like typical iron starvation genes. The protein derived from NCgl0943 is composed of 331 amino acid residues (36.044 kDa) and contains a DNA binding domain of the AraC family (PF00165 in the PFAM data base (21Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar), PS01124 in the PROSITE data base (22Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J.A. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (898) Google Scholar)) with two helix-turn-helix motifs extending from position 113 to 159 and from position 165 to 208. It is flanked by amino- and carboxyl-terminal domains of 112 and 123 residues, respectively, which show no significant sequence similarity to other proteins. Based on the results described below, the NCgl0943 gene was designated ripA (repressor of iron proteins A). In order to test an involvement of the RipA protein in acn regulation, a ripA deletion mutant of C. glutamicum was constructed. In a first set of experiments, the growth behavior of the ΔripA mutant was tested. As shown in Fig. 1A, no differences were observed between wild type and mutant cultivated in glucose minimal medium containing excess iron (100 μm). However, under iron-limiting conditions (1 μm), the ripA mutant grew initially like the wild type, but after an A600 of about 5, the growth rate of the mutant decreased more strongly than that of the wild type. The final cell density of the mutant (A600 of 20) was only half that of the wild type (A600 = 40). Thus, the ΔripA mutant has a growth defect under iron limitation but not under iron excess. As shown in Fig. 1C, this growth defect could be reversed by transformation with a plasmid carrying the ripA gene with its native promoter region (pJC1-ripA), but not with pJC1 alone (Fig. 1B). In a second set of experiments, aconitase activity was determined in wild-type and ΔripA cells from cultures grown under iron excess and iron limitation. As shown in Fig. 2, the aconitase activity of the two strains was nearly identical under iron excess, whereas under iron limitation, the ΔripA mutant had a 1.5–2-fold higher activity than the wild type at four different time points. Thus, the absence of ripA might result in an increased expression of the acn gene under iron limitation, but not under iron excess. Comparison of the Expression Profiles of ΔripA Mutant and Wild Type with DNA Chips—In order to determine the effects of RipA on acn expression as well as on global gene expression, whole genome DNA microarrays of C. glutamicum (16Wendisch V.F. J. Biotechnol. 2003; 104: 273-285Crossref PubMed Scopus (114) Google Scholar) were used to compare the mRNA ratios of the ΔripA mutant and the wild type under iron limitation and iron excess. Under iron starvation (1 μm iron), nine genes showed a >1.7-fold higher mRNA level in the ΔripA mutant (TABLE ONE). This group included the aconitase gene acn, supporting the above made assumption that increased acn expression is responsible for the elevated aconitase activity in the ΔripA mutant under iron limitation. Besides acn, catA (catechol 1,2-dioxygenase), leuCD (isopropylmalate dehydratase), narKGHJI (nitrate/nitrite transporter and nitrate reductase), sdhCAB (succinate dehydrogenase), and pta (phosphotransacetylase) showed higher mRNA levels in the ΔripA mutant compared with the wild type. The mRNA level of the ackA gene for acetate kinase, which is co-transcribed with pta (23Reinscheid D.J. Schnicke S. Rittmann D. Zahnow U. Sahm H. Eikmanns B.J. Microbiology. 1999; 145: 503-513Crossref PubMed Scopus (54) Google Scholar), was slightly increased in the ΔripA mutant but below the cut-off used. Except for the transporter NarK, phosphotransacetylase, and acetate kinase, the enzymes encoded by these genes are known to contain iron, mostly in the form of iron-sulfur clusters (aconitase, isopropylmalate dehydratase, nitrate reductase, succinate dehydrogenase) and/or heme (nitrate reductase, succinate dehydrogenase) (24Bott M. Niebisch A. J. Biotechnol. 2003; 104: 129-153Crossref PubMed Scopus (157) Google Scholar). Remarkably, the mRNA level of the genes mentioned above was changed only under iron limitation but not under iron excess (TABLE ONE).TABLE ONEGenes showing altered expression in the C. glutamicum ΔripA mutant compared with wild typeNCgl numberaThis column includes those genes whose average mRNA ratio (ΔripA mutant/wild type) was altered ≥1.7-fold or ≤1.7-fold (p value ≤0.05) in five DNA microarray experiments performed with RNA isolated from five independent cultivations in CGXII minimal medium under iron limitation (1 μm FeSO4). The genes leuC, sdhB, narK, and narH show an average mRNA ratio below 1.7 but were included, since they are organized in operons with genes (leuD, sdhCA, or narKGJI) having an mRNA ratio above 1.7GeneAnnotationRatio, iron limitationaThis column includes those genes whose average mRNA ratio (ΔripA mutant/wild type) was altered ≥1.7-fold or ≤1.7-fold (p value ≤0.05) in five DNA microarray experiments performed with RNA isolated from five independent cultivations in CGXII minimal medium under iron limitation (1 μm FeSO4). The genes leuC, sdhB, narK, and narH show an average mRNA ratio below 1.7 but were included, since they are organized in operons with genes (leuD, sdhCA, or narKGJI) having an mRNA ratio above 1.7Ratio, iron excitationbThis column provides the mRNA ratio (ΔripA mutant/wild type) of the genes under iron excess conditions. It represents the average of two DNA microarray experiments performed with RNA isolated from two independent cultivations in CGXII minimal medium under iron excess (100 μm FeSO4)NCgl2319catACatechol 1,2-dioxygenase4.411.03NCgl1482acnAconitase2.400.88NCgl1262leuC3-Isopropylmalate dehydratase, large subunit1.650.90NCgl1263leuD3-Isopropylmalate dehydratase, small subunit2.130.91NCgl0359sdhCSuccinate dehydrogenase, cytochrome b subunit1.900.85NCgl0360sdhASuccinate dehydrogenase, flavoprotein1.710.81NCgl0361sdhBSuccinate dehydrogenase, FeS protein1.641.01NCgl1143narKNitrate/nitrite transporter1.631.04NCgl1142narGNitrate reductase, α subunit1.891.00NCgl1141narHNitrate reductase, β subunit1.670.90NCgl1140narJNitrate reductase, δ subunit1.751.05NCgl1139narINitrate reductase, γ subunit1.720.99NCgl2657ptaPhosphotransacetylase1.821.24NCgl2439ftnFerritin0.550.58NCgl1490Putative membrane protein0.520.65NCgl2434Putative membrane protein0.460.91NCgl0140Putative sugar O-acetyltransferase0.441.09NCgl1096Putative flavin-containing monooxygenase0.380.46NCgl2001Conserved hypothetical protein0.300.81NCgl2897dpsStarvation-induced DNA protection protein0.290.30NCgl0943ripATranscriptional regulator, AraC family0.110.16a This column includes those genes whose average mRNA ratio (ΔripA mutant/wild type) was altered ≥1.7-fold or ≤1.7-fold (p value ≤0.05) in five DNA microarray experiments performed with RNA isolated from five independent cultivations in CGXII minimal medium under iron limitation (1 μm FeSO4). The genes leuC, sdhB, narK, and narH show an average mRNA ratio below 1.7 but were included, since they are organized in operons with genes (leuD, sdhCA, or narKGJI) having an mRNA ratio above 1.7b This column provides the mRNA ratio (ΔripA mutant/wild type) of the genes under iron excess conditions. It represents the average of t
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