Cyanobacterial FurA works as a global regulator linking iron homeostasis to photosynthetic metabolism and the responses to different environmental stresses. Additionally, FurA modulates several genes involved in redox homeostasis and fulfills the characteristics of a heme-sensor protein whose interaction with this cofactor negatively affects its DNA binding ability. FurA from Anabaena PCC 7120 contains five cysteine residues, four of them arranged in two redox CXXC motifs. Aims: Our goals were to analyze in depth the putative contribution of these CXXC motifs in the redox properties of FurA and to identify potential interacting partners of this regulator. Results: Insulin reduction assays unravel that FurA exhibits disulfide reductase activity. Simultaneous presence of both CXXC signatures greatly enhances the reduction rate, although the redox motif containing Cys101 and Cys104 seems a major contributor to this activity. Disulfide reductase activity was not detected in other ferric uptake regulator (Fur) proteins isolated from heterotrophic bacteria. In vivo, FurA presents different redox states involving intramolecular disulfide bonds when is partially oxidized. Redox potential values for CXXC motifs, −235 and −238 mV, are consistent with those reported for other proteins displaying disulfide reductase activity. Pull-down and two-hybrid assays unveil potential FurA interacting partners, namely phosphoribulokinase Alr4123, the hypothetical amidase-containing domain All1140 and the DNA-binding protein HU. Innovation: A novel biochemical activity of cyanobacterial FurA based on its cysteine arrangements and the identification of novel interacting partners are reported. Conclusion: The present study discloses a putative connection of FurA with the cyanobacterial redox-signaling pathway. Antioxid. Redox Signal. 20, 1396–1406.
Get full access to this article
View all access options for this article.
References
1.
AbateC, PatelL, RauscherFJ3rd., and CurranT. Redox regulation of Fos and Jun DNA-binding activity in vitro. Science, 249: 1157–1161, 1990.
2.
BaggA and NeilandsJB. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry, 26: 5471–5477, 1987.
3.
BattestiA and BouveretE. The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods, 58: 325–334, 2012.
4.
BenoitR and AuerM. A direct way of redox sensing. RNA Biol, 8: 18–23, 2011.
5.
BesMT, HernandezJA, PeleatoML, and FillatMF. Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter. FEMS Microbiol Lett, 194: 187–192, 2001.
6.
BuchananBB. Regulation of CO2 assimilation in oxygenic photosynthesis: the ferredoxin/thioredoxin system. Perspective on its discovery, present status, and future development. Arch Biochem Biophys, 288: 1–9, 1991.
7.
ButcherJ, SarvanS, BrunzelleJS, CoutureJF, and StintziA. Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation. Proc Natl Acad Sci U S A, 109: 10047–10052, 2012.
8.
CameronJC and PakrasiHB. Essential role of glutathione in acclimation to environmental and redox perturbations in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol, 154: 1672–1685, 2010.
9.
ChiversPT, PrehodaKE, and RainesRT. The CXXC motif: a rheostat in the active site. Biochemistry, 36: 4061–4066, 1997.
10.
DanciuTE and WhitmanM. Oxidative stress drives disulfide bond formation between basic helix-loop-helix transcription factors. J Cell Biochem, 109: 417–424, 2010.
11.
DelanyI, SpohnG, RappuoliR, and ScarlatoV. The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol Microbiol, 42: 1297–1309, 2001.
12.
DianC, VitaleS, LeonardGA, BahlawaneC, FauquantC, LeducD, MullerC, de ReuseH, Michaud-SoretI, and TerradotL. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites. Mol Microbiol, 79: 1260–1275, 2011.
13.
ErnstJF, BennettRL, and RothfieldLI. Constitutive expression of the iron-enterochelin and ferrichrome uptake systems in a mutant strain of Salmonella typhimurium. J Bacteriol, 135: 928–934, 1978.
14.
FillatMF. Fur (ferric uptake regulation) proteins in cyanobacteria. In: Biometals: Molecular Structures, Binding Properties and Applications, edited by BlancG and MoreauD. New York: Science Publishers, Inc., 2010, pp. 139–162.
15.
FioriniF, StefaniniS, ValentiP, ChianconeE, and De BiaseD. Transcription of the Listeria monocytogenes fri gene is growth-phase dependent and is repressed directly by Fur, the ferric uptake regulator. Gene, 410: 113–121, 2008.
16.
FlorencioFJ, Perez-PerezME, Lopez-MauryL, Mata-CabanaA, and LindahlM. The diversity and complexity of the cyanobacterial thioredoxin systems. Photosynth Res, 89: 157–171, 2006.
17.
FomenkoDE and GladyshevVN. Identity and functions of CxxC-derived motifs. Biochemistry, 42: 11214–11225, 2003.
18.
GaoXH, BedhommeM, VeyelD, ZaffagniniM, and LemaireSD. Methods for analysis of protein glutathionylation and their application to photosynthetic organisms. Mol Plant, 2: 218–235, 2009.
19.
GleasonFK. Thioredoxins in cyanobacteria: structure and redox regulation of enzyme activity. In: The Molecular Biology of Cyanobacteria. Advances in Photosynthesis and Respiration, edited by BryantDA. Netherlands: Springer, 2004, pp. 715–729.
20.
GonzalezA, BesMT, BarjaF, PeleatoML, and FillatMF. Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology. Plant Cell Physiol, 51: 1900–1914, 2010.
21.
GonzalezA, BesMT, PeleatoML, and FillatMF. Unravelling the regulatory function of FurA in Anabaena sp. PCC 7120 through 2-D DIGE proteomic analysis. J Proteomics, 74: 660–671, 2011.
22.
GonzalezA, BesMT, ValladaresA, PeleatoML, and FillatMF. FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120. Environ Microbiol, 14: 3175–3187, 2012.
23.
HagiwaraM, MaegawaK, SuzukiM, UshiodaR, ArakiK, MatsumotoY, HosekiJ, NagataK, and InabaK. Structural basis of an ERAD pathway mediated by the ER-resident protein disulfide reductase ERdj5. Mol Cell, 41: 432–444, 2011.
24.
HerasB, EdelingMA, ByrielKA, JonesA, RainaS, and MartinJL. Dehydration converts DsbG crystal diffraction from low to high resolution. Structure, 11: 139–145, 2003.
25.
HerasB, KurzM, ShouldiceSR, and MartinJL. The name's bond …… disulfide bond. Curr Opin Struct Biol, 17: 691–698, 2007.
26.
HernandezJA, BesMT, FillatMF, NeiraJL, and PeleatoML. Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state. Biochem J, 366: 315–322, 2002.
27.
HernandezJA, Lopez-GomollonS, Muro-PastorA, ValladaresA, BesMT, PeleatoML, and FillatMF. Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn(2+) are positive effectors in the binding to isiB and furA promoters. Biometals, 19: 259–268, 2006.
28.
HolmgrenA. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem, 254: 9627–9632, 1979.
29.
HolmgrenA. Thioredoxin. Annu Rev Biochem, 54: 237–271, 1985.
30.
HoriuchiM, NakamuraK, KojimaK, NishiyamaY, HatakeyamaW, HisaboriT, and HiharaY. The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria. Biochem J, 431: 135–140, 2010.
31.
InabaK, TakahashiYH, and ItoK. Reactivities of quinone-free DsbB from Escherichia coli. J Biol Chem, 280: 33035–33044, 2005.
32.
JozefczakM, RemansT, VangronsveldJ, and CuypersA. Glutathione is a key player in metal-induced oxidative stress defenses. Int J Mol Sci, 13: 3145–3175, 2012.
33.
KarimovaG, PidouxJ, UllmannA, and LadantD. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A, 95: 5752–5756, 1998.
34.
KatsubeT, MatsumotoS, TakatsukaM, OkuyamaM, OzekiY, NaitoM, NishiuchiY, FujiwaraN, YoshimuraM, TsuboiT, ToriiM, OshitaniN, ArakawaT, and KobayashiK. Control of cell wall assembly by a histone-like protein in Mycobacteria. J Bacteriol, 189: 8241–8249, 2007.
35.
KhudyakovI and WolkCP. Evidence that the hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp. strain PCC 7120. J Bacteriol, 178: 3572–3577, 1996.
36.
KobayashiD, TamoiM, IwakiT, ShigeokaS, and WadanoA. Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942. Plant Cell Physiol, 44: 269–276, 2003.
37.
KojimaK, MotohashiK, MorotaT, OshitaM, HisaboriT, HayashiH, and NishiyamaY. Regulation of translation by the redox state of elongation factor G in the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem, 284: 18685–18691, 2009.
38.
LaemmliUK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–685, 1970.
39.
LatifiA, RuizM, and ZhangCC. Oxidative stress in cyanobacteria. FEMS Microbiol Rev, 33: 258–278, 2009.
40.
LeichertLI and JakobU. Global methods to monitor the thiol-disulfide state of proteins in vivo. Antioxid Redox Signal, 8: 763–772, 2006.
41.
Lopez-GomollonS, HernandezJA, PellicerS, AngaricaVE, PeleatoML, and FillatMF. Cross-talk between iron and nitrogen regulatory networks in Anabaena (Nostoc) sp. PCC 7120: identification of overlapping genes in FurA and NtcA regulons. J Mol Biol, 374: 267–281, 2007.
42.
Lopez-GomollonS, HernandezJA, WolkCP, PeleatoML, and FillatMF. Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp. PCC 7120. Microbiology, 153: 42–50, 2007.
43.
López-GomollónS, SevillaE, BesMT, PeleatoML and FillatMF. New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress. Biochem J, 418: 201–207, 2009.
44.
Martinez-GalisteoE, PadillaCA, Garcia-AlfonsoC, Lopez-BareaJ, and BarcenaJA. Purification and properties of bovine thioredoxin system. Biochimie, 75: 803–809, 1993.
45.
Mata-CabanaA, Garcia-DominguezM, FlorencioFJ, and LindahlM. Thiol-based redox modulation of a cyanobacterial eukaryotic-type serine/threonine kinase required for oxidative stress tolerance. Antioxid Redox Signal, 17: 521–533, 2007.
46.
MoslavacS, BredemeierR, MirusO, GranvoglB, EichackerLA, and SchleiffE. Proteomic analysis of the outer membrane of Anabaena sp. strain PCC 7120. J Proteome Res, 4: 1330–1338, 2005.
47.
NagarajaR and HaselkornR. Protein HU from the cyanobacterium Anabaena. Biochimie, 76: 1082–1089, 1994.
48.
PellicerS, BesMT, GonzalezA, NeiraJL, PeleatoML, and FillatMF. High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment. Process Biochem, 45: 292–296, 2010.
49.
PellicerS, GonzalezA, PeleatoML, MartinezJI, FillatMF, and BesMT. Site-directed mutagenesis and spectral studies suggest a putative role of FurA from Anabaena sp. PCC 7120 as a heme sensor protein. FEBS J, 279: 2231–2246, 2012.
50.
Perez-PerezME, Mata-CabanaA, Sanchez-RiegoAM, LindahlM, and FlorencioFJ. A comprehensive analysis of the peroxiredoxin reduction system in the Cyanobacterium Synechocystis sp. strain PCC 6803 reveals that all five peroxiredoxins are thioredoxin dependent. J Bacteriol, 191: 7477–7489, 2009.
51.
QuanS, SchneiderI, PanJ, Von HachtA, and BardwellJC. The CXXC motif is more than a redox rheostat. J Biol Chem, 282: 28823–28833, 2007.
52.
Rasband. ImageJ, U. S. National Institutes of Health, Bethesda, Maryland. http://imagej.nih.gov/ij/, 1997–2012.
53.
RippkaR, DeruellesJ, WaterburyJB, HerdmanM, and StanierRY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol, 111: 1–61, 1979.
54.
SevierCS and KaiserCA. Formation and transfer of disulphide bonds in living cells. Nat Rev Mol Cell Biol, 3: 836–847, 2002.
55.
ShcolnickS and KerenN. Metal homeostasis in cyanobacteria and chloroplasts. Balancing benefits and risks to the photosynthetic apparatus. Plant Physiol, 141: 805–810, 2006.
56.
SinghAK, Bhattacharyya-PakrasiM, and PakrasiHB. Identification of an atypical membrane protein involved in the formation of protein disulfide bonds in oxygenic photosynthetic organisms. J Biol Chem, 283: 15762–15770, 2008.
57.
StudierFW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif, 41: 207–234, 2005.
58.
TouatiD. Iron and oxidative stress in bacteria. Arch Biochem Biophys, 373: 1–6, 2000.
59.
van VlietAH, StoofJ, PoppelaarsSW, BereswillS, HomuthG, KistM, KuipersEJ, and KustersJG. Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori Fur repressor. J Biol Chem, 278: 9052–9057, 2003.
WatnickPI, EtoT, TakahashiH, and CalderwoodSB. Purification of Vibrio cholerae Fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay. J Bacteriol, 179: 243–247, 1997.
62.
WoutersMA, FanSW, and HaworthNL. Disulfides as redox switches: from molecular mechanisms to functional significance. Antioxid Redox Signal, 12: 53–91, 2010.
63.
YoshimuraH, IkeuchiM, and OhomoriM. Cell surface-associated proteins in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Microbes Environ, 27: 538–543, 2012.
64.
ZhaoK, BaiW, and MiH. Dielectric spectroscopy of Anabaena 7120 protoplast suspensions. Bioelectrochemistry, 69: 49–57, 2006.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
