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Genes  2011 

Proteomics Analysis of the Effects of Cyanate on Chromobacterium violaceum Metabolism

DOI: 10.3390/genes2040736

Keywords: 2DE, bacteria, cyanate, mass spectrometry, proteomic

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Abstract:

Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium. The proteome of cells grown with and without cyanate was compared on 2-D gels. Differential spots were digested and identified by mass spectrometry. The bacterium was able to grow at concentrations of up to 1 mM cyanate. Eighteen spots were differentially expressed in the presence of cyanate, of which 16 were downregulated and only two were upregulated. An additional 12 spots were detected only in extracts of cells unexposed to cyanate, and one was expressed only by the exposed cells. Fourteen spots were identified, corresponding to 13 different proteins. We conclude that cyanate promotes expression of enzymes that combat oxidative stress and represses enzymes of the citric acid cycle, strongly affecting the energetic metabolism of the cell. Other proteins that were under-expressed in bacteria exposed to cyanate are involved in amino-acid metabolism or are hypothetical proteins, demonstrating that cyanate also affects expression of genes that are not part of the cyn operon.

References

[1]  Hungria, M.; Astolfi-Filho, S.; Chueire, L.M.O.; Nicolás, M.F.; Santos, E.B.P.; Bulbol, M.R.; Souza-Filho, A.; Nogueira-Assun??o, E.; Germano, M.G.; Vasconcelos, A.R.T. Genetic characterization of Chromobacterium isolates from Black water environments in the Brazilian Amazon. Lett. Appl. Microbiol. 2005, 41, 17–23.
[2]  Dall'Agnol, L.T.; Martins, R.N.; Vallinoto, A.C.R.; Ribeiro, K.S.T. Diversity of Chromobacterium violaceum isolates from aquatic environments of state of Pará, Brazilian Amazon. Mem. Inst. Oswaldo Cruz 2008, 103, 678–682.
[3]  Lima-Bittencourt, C.I.; Astolfi-Filho, S.; Chartone-Souza, E.; Santos, F.R.; Nascimento, A.M.A. Analysis of Chromobacterium sp. natural isolates from different Brazilian ecosystems. BMC Microbiol. 2007, 7, 58.
[4]  Hungria, M.; Nicolás, M.F.; Guimar?es, C.T.; Vasconcelos, A.T.R. Tolerance to stress and environmental adaptability of Chromobacterium violaceum. Genet. Mol. Res. 2004, 3, 102–116.
[5]  Brazilian Genome Consortium. The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. USA 2003, 100, 11660–11665.
[6]  Carepo, M.S.P.; Azevedo, J.S.N.; Porto, J.I.R.; Bentes-Souza, A.R.; Batista, J.S.; Silva, A.L.C.; Schneider, M.P.C. Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification. Genet. Mol. Res. 2004, 3, 181–194.
[7]  Lamblin, A.F.; Fuchs, J.A. Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation. J. Bacteriol. 1994, 176, 6613–6622.
[8]  Walker, J.; Hambly, F.J. Transformation of ammonium cyanate into urea. J. Chem. Soc. 1895, 67, 746–767.
[9]  Baxter, J.; Cummings, S.P. The current and future applications of microorganism in the bioremediation of cyanide contamination. Antonie Van Leeuwenhoek 2006, 90, 1–17.
[10]  Stark, G.R. Reactions of cyanate with functional groups of proteins. III. Reactions with amino and carboxyl groups. Biochemistry 1965, 4, 1030–1036.
[11]  Srivastava, V.K.; Varshney, N.; Jaiswal, A. In vivo effect of cyanate on serum and eye lens in rat. Indian J. Exp. Biol. 1993, 31, 83–84.
[12]  Kamennaya, N.A.; Chernihovsky, M.; Post, A.F. The cyanate utilization capacity of marine unicellular Cyanobacteria. Limnol. Oceanogr. 2008, 53, 2485–2494.
[13]  Luque-Almagro, V.M.; Huertas, M.J.; Saéz, L.P.; Luque-Romero, M.M.; Moreno-Vivián, C.; Castillo, F.; Roldán, M.D.; Blasco, R. Characterization of the Pseudomonas pseudoalcaligenes CECT5344 cyanase, an enzyme that is not essential for cyanide assimilation. Appl. Environ. Microbiol. 2008, 74, 6280–6288.
[14]  Espie, G.S.; Jalali, F.; Tong, T.; Zacal, N.J.; So, A.K.C. Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by Cyanobacteria. J. Bacteriol. 2007, 189, 1013–1024.
[15]  Miyamoto, K.; Kosakai, K.; Ikebayashi, S.; Tsuchiya, T.; Yamamoto, S.; Tsujibo, H. Proteomic analysis of Vibrio vulnificus M2799 grown under iron-repleted and iron-depleted conditions. Microb. Pathog. 2009, 46, 171–177.
[16]  Roma-Rodrigues, C.; Santos, P.M.; Benndorf, D.; Rapp, E.; Sá-Correia, I. Response of Pseudomonas putida KT2440 to phenol at the level of membrane proteome. J. Proteomics 2010, 73, 1461–1478.
[17]  Vintila, S.; Jonasson, S.; Wadensten, H.; Nilsson, A.; Andrén, P.E.; El-Shehawy, R. Proteomic profiling of the Baltic Sea cyanobacterium Nodularia spumigena strain AV1 during ammonium supplementation. J. Proteomics 2010, 73, 1670–1679.
[18]  Cacace, G.; Mazzeo, M.F.; Sorrentino, A.; Spada, V.; Malorni, A.; Siciliano, R.A. Proteomics for elucidation of cold adaptation mechanisms in Listeria monocytogenes. J. Proteomics 2010, 73, 2021–2030.
[19]  Boening, D.W.; Chew, C.M. A critical review: General toxicity and environmental fate of three aqueous cyanide ions associated ligands. Water Air Soil Pollut. 1999, 109, 67–79.
[20]  Guilloton, M.; Karst, F. Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: A case of by-product inhibition? J. Gen. MIcrobiol. 1987, 133, 655–665.
[21]  Kunz, D.A.; Nagappan, O. Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764. Appl. Environ. Microbiol. 1989, 55, 256–258.
[22]  Bhasin, M.; Garg, A.; Raghava, G.P.S. PSLpred: Prediction of subcellular localization of bacterial proteins. Bioinformatics 2005, 21, 2522–2524.
[23]  Yu, N.Y.; Wagner, J.R.; Laird, M.R.; Melli, G.; Rey, S.; Lo, R.; Dao, P.; Sahinalp, S.C.; Ester, M.; Foster, L.J.; et al. PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010, 26, 1608–1615.
[24]  Hiller, K.; Grote, A.; Maneck, M.; Münch, R.; Jahn, D. JVirGel 2.0: Computational prediction of proteomes separated via two-dimensional gel electrophoresis under consideration of membrane and secreted proteins. Bioinformatics 2006, 22, 2441–2443.
[25]  Castellana, N.; Bafna, V. Proteogenomics to discover the full coding content of genomes: A computational perspective. J. Proteomics 2010, 73, 2124–2135.
[26]  Batista, J.S.S.; Torres, A.R.; Hungria, M. Towards a two-dimensional proteomic reference map of Bradyrhizobium japonicum CPAC 15: Spotlighting “hypothetical proteins”. Proteomics 2010, 10, 3176–3189.
[27]  Quevillon, E.; Silventoinen, V.; Pillai, S.; Harte, N.; Mulder, N.; Apweiler, R.; Lopez, R. InterProScan: Proteins domain identifier. Nucleic Acids Res. 2005, 33, W116–W120.
[28]  Poulsen, P.; Jensen, K.F. Three genes preceding pyrE on the Escherichia coli chromosome are essential for survival and normal cell morphology in stationary culture and at high temperature. Res. Micrbiol. 1991, 142, 283–288.
[29]  Ksi??ek, K. Bacterial aging: From mechanistic basis to evolutionary perspective. Cell. Mol. Life Sci. 2010, 67, 3131–3137.
[30]  Srivastava, A.K.; Bhargava, P.; Thapar, R.; Rai, L.C. Differential response of antioxidative defense system of Anabaena doliolum under arsenite and arsenate stress. J. Basic Microbiol. 2009, 49, S63–S72.
[31]  Dukan, S.; Nystr?m, T. Bacterial senescence: Stasis results in increased and differential oxidation of cytoplasmic proteins leading to developmental induction of the heat shock regulon. Genes Dev. 1998, 12, 3431–3441.
[32]  Michaels, R.; Corpe, W.A. Cyanide formation by Chromobacterium violaceum. J. Bacteriol. 1965, 89, 106–112.
[33]  Rodgers, P.B.; Knowles, C.J. Cyanide production and degradation during growth of Chromobacterium violaceum. J. Gen. Microbiol. 1978, 108, 261–267.
[34]  Petrak, J.; Ivanek, R.; Toman, O.; Cmejla, R.; Cmejlova, J.; Vyoral, D.; Zivny, J.; Vulpe, C.D. Déjà vu in proteomics. A hit parade of repeatedly indentified differentially expressed proteins. Proteomics 2008, 8, 1744–1749.
[35]  Wang, P.; Bouwman, F.G.; Mariman, E.C.M. Generally detected proteins in comparative proteomics—A matter of cellular stress response? Proteomics 2009, 9, 2955–2966.
[36]  Hansen, H.M.; Lehnherr, H.; Wang, X.; Mobley, V.; Jin, D.J. Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes. Mol. Microbiol. 2003, 48, 1621–1631.
[37]  Williams, M.D.; Ouyang, T.X.; Flickinger, M.C. Starvation-induced expression of SspA and SspB: The effects of a null mutation in SspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation. Mol. Microbiol. 1994, 11, 1029–1043.
[38]  Durmowicz, M.C.; Maier, R.J. Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum. J. Bacteriol. 1997, 179, 3676–3682.
[39]  Buhrke, T.; Friedrich, B. hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster. Arch. Microbiol. 1998, 170, 460–463.
[40]  Szklarczyk, D.; Franceschini, A.; Kuhn, M.; Simonovic, M.; Roth, A.; Minguez, P.; Doerks, T.; Stark, M.; Muller, J.; Bork, P.; et al. The STRING database in 2011: Functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011, 39, D561–D568.
[41]  Creczynski-Pasa, T.B.; Ant?nio, R.V. Energetic metabolism of Chromobacterium violaceum. Genet. Mol. Res. 2004, 3, 162–166.
[42]  Dowsey, A.W.; English, J.A.; Lisacek, F.; Morris, J.S.; Yang, G.Z.; Dunn, M.J. Image analysis tools and emerging algorithms for expression proteomics. Proteomics 2010, 10, 4226–4257.
[43]  Perkins, D.N.; Pappin, D.J.C.; Creasy, D.M.; Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567.
[44]  Tatusov, R.L.; Fedorova, N.D.; Jackson, J.D.; Jacobs, A.R.; Kiryutin, B.; Koonin, E.V.; Krylov, D.M.; Mazumder, R.; Mekhedov, S.L.; Nikolskaya, A.N.; et al. The COG database: An updated version includes eukaryotes. BMC Bioinformatics 2003, 4, 41.
[45]  Farmazi, M.A.; Stagars, M.; Pensini, E.; Krebs, W.; Brandi, H. Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterium violaceum. J. Biotechnol. 2004, 113, 321–326.
[46]  Akcil, A. Destruction of cyanide in gold mill effluents: Biological versus chemical treatments. Biotechnol. Adv. 2003, 21, 501–511.

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