We characterized a new quorum-sensing regulator, PlcRa, which is present in various members of the B. cereus group and identified a signaling heptapeptide for PlcRa activity: PapRa7. We demonstrated that PlcRa is a 3D structural paralog of PlcR using sequence analysis and homology modeling. A comparison of the transcriptomes at the onset of stationary phase of a ΔplcRa mutant and the wild-type B. cereus ATCC 14579 strain showed that 68 genes were upregulated and 49 genes were downregulated in the ΔplcRa mutant strain (>3-fold change). Genes involved in the cysteine metabolism (putative CymR regulon) were downregulated in the ΔplcRa mutant strain. We focused on the gene with the largest difference in expression level between the two conditions, which encoded -AbrB2- a new regulator of the AbrB family. We demonstrated that purified PlcRa bound specifically to the abrB2 promoter in the presence of synthetic PapRa7, in an electrophoretic mobility shift assay. We further showed that the AbrB2 regulator controlled the expression of the yrrT operon involved in methionine to cysteine conversion. We found that the ΔplcRa mutant strain was more sensitive to hydrogen peroxide- and disulfide-induced stresses than the wild type. When cystine was added to the culture of the ΔplcRa mutant, challenged with hydrogen peroxide, growth inhibition was abolished. In conclusion, we identified a new RNPP transcriptional regulator in B. cereus that activated the oxidative stress response and cysteine metabolism in transition state cells.
References
[1]
Stenfors Arnesen LP, Fagerlund A, Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32: 579–606.
Phillips ZE, Strauch MA (2002) Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59: 392–402.
[4]
Price CW, Fawcett P, Ceremonie H, Su N, Murphy CK, et al. (2001) Genome-wide analysis of the general stress response in Bacillus subtilis. Mol Microbiol 41: 757–774.
[5]
Zuber P (2009) Management of oxidative stress in Bacillus. Annu Rev Microbiol 63: 575–597.
[6]
Dunny GM, Leonard BA (1997) Cell-cell communication in gram-positive bacteria. Annu Rev Microbiol 51: 527–564.
[7]
Gohar M, Faegri K, Perchat S, Ravnum S, Okstad OA, et al. (2008) The PlcR virulence regulon of Bacillus cereus. PLoS One 3: e2793.
[8]
Agaisse H, Gominet M, Okstad OA, Kolsto AB, Lereclus D (1999) PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol Microbiol 32: 1043–1053.
[9]
Bouillaut L, Perchat S, Arold S, Zorrilla S, Slamti L, et al. (2008) Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides. Nucleic Acids Res 36: 3791–3801.
[10]
Gominet M, Slamti L, Gilois N, Rose M, Lereclus D (2001) Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol Microbiol 40: 963–975.
[11]
Slamti L, Lereclus D (2002) A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J 21: 4550–4559.
[12]
Declerck N, Bouillaut L, Chaix D, Rugani N, Slamti L, et al. (2007) Structure of PlcR: Insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria. Proc Natl Acad Sci U S A 104: 18490–18495.
[13]
D'Andrea LD, Regan L (2003) TPR proteins: the versatile helix. Trends Biochem Sci 28: 655–662.
[14]
Perchat S, Dubois T, Zouhir S, Gominet M, Poncet S, et al. (2011) A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group. Mol Microbiol 82: 619–633.
[15]
Shi K, Brown CK, Gu ZY, Kozlowicz BK, Dunny GM, et al. (2005) Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis. Proc Natl Acad Sci U S A 102: 18596–18601.
[16]
Solomon JM, Lazazzera BA, Grossman AD (1996) Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev 10: 2014–2024.
[17]
Parashar V, Mirouze N, Dubnau DA, Neiditch MB (2011) Structural basis of response regulator dephosphorylation by Rap phosphatases. PLoS Biol 9: e1000589.
[18]
Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, et al. (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423: 87–91.
[19]
Ross CL, Koehler TM (2006) plcR papR-independent expression of anthrolysin O by Bacillus anthracis.. J Bacteriol 188: 7823–7829.
[20]
Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234: 779–815.
[21]
Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2: 953–971.
[22]
Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, et al (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30: 1545–1614.
[23]
Agaisse H, Lereclus D (1994) Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis. Mol Microbiol 13: 97–107.
[24]
Moran CP (1993) RNA polymerase and transcription factors. In: Sonenshein AL, Hoch, J.A and Losick, R., editor. Bacillus subtilis and other gram-positive bacteria. Washington D.C.: American Society for Microbiology. 653–667.
[25]
Ceragioli M, Mols M, Moezelaar R, Ghelardi E, Senesi S, et al. (2010) Comparative transcriptomic and phenotypic analysis of the responses of Bacillus cereus to various disinfectant treatments. Appl Environ Microbiol 76: 3352–3360.
[26]
Mols M, Abee T (2011) Primary and secondary oxidative stress in Bacillus. Environ Microbiol 13: 1387–1394.
[27]
Tu WY, Pohl S, Gizynski K, Harwood CR (2012) The iron-binding protein Dps2 confers peroxide stress resistance on Bacillus anthracis. J Bacteriol 194: 925–931.
[28]
van Schaik W, van der Voort M, Molenaar D, Moezelaar R, de Vos WM, et al. (2007) Identification of the sigmaB regulon of Bacillus cereus and conservation of sigmaB-regulated genes in low-GC-content gram-positive bacteria. J Bacteriol 189: 4384–4390.
[29]
Smeesters PR, Dreze PA, Bousbata S, Parikka KJ, Timmery S, et al. (2011) Characterization of a novel temperate phage originating from a cereulide-producing Bacillus cereus strain. Res Microbiol 162: 446–459.
[30]
Graumann PL, Knust T (2009) Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair. Chromosome Res 17: 265–275.
[31]
Kolsto AB, Tourasse NJ, Okstad OA (2009) What sets Bacillus anthracis apart from other Bacillus species? Annu Rev Microbiol 63: 451–476.
[32]
Salvetti S, Faegri K, Ghelardi E, Kolsto AB, Senesi S (2011) Global Gene Expression Profile for Swarming Bacillus cereus Bacteria. Appl Environ Microbiol 77: 5149–5156.
[33]
Burguiere P, Auger S, Hullo MF, Danchin A, Martin-Verstraete I (2004) Three different systems participate in L-cystine uptake in Bacillus subtilis. J Bacteriol 186: 4875–4884.
[34]
Hullo MF, Auger S, Soutourina O, Barzu O, Yvon M, et al. (2007) Conversion of methionine to cysteine in Bacillus subtilis and its regulation. J Bacteriol 189: 187–197.
[35]
Even S, Burguiere P, Auger S, Soutourina O, Danchin A, et al. (2006) Global control of cysteine metabolism by CymR in Bacillus subtilis. J Bacteriol 188: 2184–2197.
[36]
Tanous C, Soutourina O, Raynal B, Hullo MF, Mervelet P, et al. (2008) The CymR regulator in complex with the enzyme CysK controls cysteine metabolism in Bacillus subtilis. J Biol Chem 283: 35551–35560.
[37]
Choi SY, Reyes D, Leelakriangsak M, Zuber P (2006) The global regulator Spx functions in the control of organosulfur metabolism in Bacillus subtilis. J Bacteriol 188: 5741–5751.
[38]
Strauch MA, Spiegelman GB, Perego M, Johnson WC, Burbulys D, et al. (1989) The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J 8: 1615–1621.
[39]
Hadjifrangiskou M, Chen Y, Koehler TM (2007) The alternative sigma factor sigmaH is required for toxin gene expression by Bacillus anthracis. J Bacteriol 189: 1874–1883.
[40]
Soutourina O, Dubrac S, Poupel O, Msadek T, Martin-Verstraete I (2010) The pleiotropic CymR regulator of Staphylococcus aureus plays an important role in virulence and stress response. PLoS Pathog 6: e1000894.
[41]
Newton GL, Rawat M, La Clair JJ, Jothivasan VK, Budiarto T, et al. (2009) Bacillithiol is an antioxidant thiol produced in Bacilli. Nat Chem Biol 5: 625–627.
[42]
Nicely NI, Parsonage D, Paige C, Newton GL, Fahey RC, et al. (2007) Structure of the type III pantothenate kinase from Bacillus anthracis at 2.0 A resolution: implications for coenzyme A-dependent redox biology. Biochemistry 46: 3234–3245.
[43]
Paige C, Reid SD, Hanna PC, Claiborne A (2008) The type III pantothenate kinase encoded by coaX is essential for growth of Bacillus anthracis. J Bacteriol 190: 6271–6275.
[44]
Auger S, Danchin A, Martin-Verstraete I (2002) Global expression profile of Bacillus subtilis grown in the presence of sulfate or methionine. J Bacteriol 184: 5179–5186.
[45]
Pohl S, Tu WY, Aldridge PD, Gillespie C, Hahne H, et al. (2011) Combined proteomic and transcriptomic analysis of the response of Bacillus anthracis to oxidative stress. Proteomics 11: 3036–3055.
[46]
Wang L, Chong H, Jiang R (2012) Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579. Appl Microbiol Biotechnol.
[47]
Hullo MF, Martin-Verstraete I, Soutourina O (2010) Complex phenotypes of a mutant inactivated for CymR, the global regulator of cysteine metabolism in Bacillus subtilis.. FEMS Microbiol Lett 309: 201–207.
[48]
Hochgrafe F, Mostertz J, Pother DC, Becher D, Helmann JD, et al. (2007) S-cysteinylation is a general mechanism for thiol protection of Bacillus subtilis proteins after oxidative stress. J Biol Chem 282: 25981–25985.
[49]
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16: 6127–6145.
[50]
Lereclus D, Arantes O, Chaufaux J, Lecadet M (1989) Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 51: 211–217.
[51]
Lereclus D, Vallade M, Chaufaux J, Arantes O, Rambaud S (1992) Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination. Biotechnology (N Y) 10: 418–421.
[52]
Sanchis V, Agaisse H, Chaufaux J, Lereclus D (1996) Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector. J Biotechnol 48: 81–96.
[53]
Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115–119.
[54]
Hsiao A, Ideker T, Olefsky JM, Subramaniam S (2005) VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data. Nucleic Acids Res 33: W627–632.
[55]
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622.
