Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR

BMC Microbiology

Catherine Duport¹  Yves Jouanneau²  Julia Esbelin¹ 

[1] INRA, UMR408, Sécurité et Qualité des Produits d’Origine Végétale, F-84914, Avignon, France;UJF-Grenoble 1/CNRS, UMR 5249, F-38041, Grenoble, France

关键词: DNA binding;    enterotoxin;    Bacillus cereus;    anaerobiosis;    Fe-S cluster;    Fnr;   

Others  :  1221846
DOI  :  10.1186/1471-2180-12-125

 received in 2012-04-06, accepted in 2012-06-11,  发布年份 2012

【 摘 要 】

Background

Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr.

Results

Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]²⁺ cluster per monomer. Atmospheric O₂ causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex.

Conclusions

Overall, this work shows that incorporation of the [4Fe-4S]²⁺ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

【 授权许可】

   
2012 Esbelin et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Clair G, Roussi S, Armengaud J, Duport C: Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions. Mol Cell Proteomics 2010, 9(7):1486-1498.
• [2]Stenfors Arnesen LP, Fagerlund A, Granum PE: From soil to gut:Bacillus cereusand its food poisoning toxins. FEMS Microbiol Rev 2008, 32(4):579-606.
• [3]Gohar M, Faegri K, Perchat S, Ravnum S, Okstad OA, Gominet M, Kolsto AB, Lereclus D: The PlcR virulence regulon of Bacillus cereus. PLoS One 2008, 3(7):e2793.
• [4]Duport C, Zigha A, Rosenfeld E, Schmitt P: Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system. J Bacteriol 2006, 188(18):6640-6651.
• [5]Zigha A, Rosenfeld E, Schmitt P, Duport C: The redox regulator Fnr is required for fermentative growth and enterotoxin synthesis in Bacillus cereus F4430/73. J Bacteriol 2007, 189(7):2813-2824.
• [6]Korner H, Sofia HJ, Zumft WG: Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol Rev 2003, 27(5):559-592.
• [7]Gruner I, Fradrich C, Bottger LH, Trautwein AX, Jahn D, Hartig E: Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4 S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr. J Biol Chem 2011, 286(3):2017-2021.
• [8]Reents H, Gruner I, Harmening U, Bottger LH, Layer G, Heathcote P, Trautwein AX, Jahn D, Hartig E: Bacillus subtilis Fnr senses oxygen via a [4Fe-4 S] cluster coordinated by three cysteine residues without change in the oligomeric state. Mol Microbiol 2006, 60(6):1432-1445.
• [9]Esbelin J, Jouanneau Y, Armengaud J, Duport C: ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus. J Bacteriol 2008, 190(12):4242-4251.
• [10]Crack J, Green J, Thomson AJ: Mechanism of oxygen sensing by the bacterial transcription factor fumarate-nitrate reduction (FNR). J Biol Chem 2004, 279(10):9278-9286.
• [11]Esbelin J, Armengaud J, Zigha A, Duport C: ResDE-dependent regulation of enterotoxin gene expression in Bacillus cereus: evidence for multiple modes of binding for ResD and interaction with Fnr. J Bacteriol 2009, 191(13):4419-4426.
• [12]Slamti L, Lereclus D: A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J 2002, 21(17):4550-4559.
• [13]Clair G, Armengaud J, Duport C: Restricting fermentative potential by proteome remodeling. Mol Cell Proteomics: an adaptive strategy evidenced in Bacillus cereus; 2012.
• [14]Reents H, Munch R, Dammeyer T, Jahn D, Hartig E: The Fnr regulon of Bacillus subtilis. J Bacteriol 2006, 188(3):1103-1112.
• [15]Jervis AJ, Crack JC, White G, Artymiuk PJ, Cheesman MR, Thomson AJ, Le Brun NE, Green J: The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4 S] to [2Fe-2 S] conversion. Proc Natl Acad Sci U S A 2009, 106(12):4659-4664.
• [16]Crack JC, den Hengst CD, Jakimowicz P, Subramanian S, Johnson MK, Buttner MJ, Thomson AJ, Le Brun NE: Characterization of [4Fe-4 S]-containing and cluster-free forms of Streptomyces WhiD. Biochemistry 2009, 48(51):12252-12264.
• [17]Dey A, Jenney FE: Adams MW, Babini E, Takahashi Y, Fukuyama K, Hodgson KO, Hedman B, Solomon EI: Solvent tuning of electrochemical potentials in the active sites of HiPIP versus ferredoxin. Science 2007, 318(5855):1464-1468.
• [18]Grzyb J, Xu F, Weiner L, Reijerse EJ, Lubitz W, Nanda V, Noy D: De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core. Biochim Biophys Acta 2010, 1797(3):406-413.
• [19]Amrein KE, Takacs B, Stieger M, Molnos J, Flint NA, Burn P: Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc Natl Acad Sci U S A 1995, 92(4):1048-1052.
• [20]Mihara H, Kurihara T, Yoshimura T, Esaki N: Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions. J Biochem 2000, 127(4):559-567.
• [21]Pelley JW, Garner CW, Little GH: A simple rapid biuret method for the estimation of protein in samples containing thiols. Anal Biochem 1978, 86(1):341-343.
• [22]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227(5259):680-685.
• [23]Blair D, Diehl H: Bathophenanthroline disulphonic acid and bathocuproine disulphonic acid, water soluble reagents for iron and copper. Talanta 1961, 7:163-174.
• [24]Pomerantsev AP, Pomerantseva OM, Leppla SH: A spontaneous translational fusion of Bacillus cereus PlcR and PapR activates transcription of PlcR-dependent genes in Bacillus anthracis via binding with a specific palindromic sequence. Infect Immun 2004, 72(10):5814-5823.