Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance.
References
[1]
Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, et al. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66–74. doi: 10.1126/science.1093857
[2]
Giebel HA, Kalhoefer D, Lemke A, Thole S, Gahl-Janssen R, et al. (2011) Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate. ISME J 5: 8–19. doi: 10.1038/ismej.2010.87
[3]
Wagner-Dobler I, Biebl H (2006) Environmental biology of the marine Roseobacter lineage. Annu Rev Microbiol 60: 255–280. doi: 10.1146/annurev.micro.60.080805.142115
Armengaud J (2013) Microbiology and proteomics, getting the best of both worlds! Environ Microbiol. 15: 12–23. doi: 10.1111/j.1462-2920.2012.02811.x
[6]
Armengaud J, Hartmann EM, Bland C (2013) Proteogenomics for environmental microbiology. Proteomics 13: 2731–42. doi: 10.1002/pmic.201200576
[7]
Christie-Oleza JA, Fernandez B, Nogales B, Bosch R, Armengaud J (2012) Proteomic insights into the lifestyle of an environmentally relevant marine bacterium. ISME J 6: 124–135. doi: 10.1038/ismej.2011.86
[8]
Clair G, Armengaud J, Duport C (2012) Restricting fermentative potential by proteome remodeling: an adaptive strategy evidenced in Bacillus cereus. Mol Cell Proteomics 11: M111 013102.
[9]
Armengaud J, Christie-Oleza JA, Clair G, Malard V, Duport C (2012) Exoproteomics: exploring the world around biological systems. Expert Rev Proteomics 9: 561–575. doi: 10.1586/epr.12.52
[10]
Christie-Oleza JA, Armengaud J (2010) In-depth analysis of exoproteomes from marine bacteria by shotgun liquid chromatography-tandem mass spectrometry: the Ruegeria pomeroyi DSS-3 case-study. Mar Drugs 8: 2223–2239. doi: 10.3390/md8082223
[11]
Clair G, Roussi S, Armengaud J, Duport C (2010) Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions. Mol Cell Proteomics 9: 1486–1498. doi: 10.1074/mcp.m000027-mcp201
[12]
Christie-Oleza JA, Pina-Villalonga JM, Bosch R, Nogales B, Armengaud J (2012) Comparative proteogenomics of twelve roseobacter exoproteomes reveals different adaptive strategies among these marine bacteria. Mol Cell Proteomics 11: M111 013110.
[13]
Chagnot C, Zorgani MA, Astruc T, Desvaux M (2013) Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective. Front Microbiol 4: 303. doi: 10.3389/fmicb.2013.00303
[14]
Wohlbrand L, Trautwein K, Rabus R (2013) Proteomic tools for environmental microbiology-a roadmap from sample preparation to protein identification and quantification. Proteomics 13: 2700–2730. doi: 10.1002/pmic.201300175
[15]
Martens T, Heidorn T, Pukall R, Simon M, Tindall BJ, et al. (2006) Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera Roseobacter, Ruegeria and Leisingera. Int J Syst Evol Microbiol 56: 1293–1304. doi: 10.1099/ijs.0.63724-0
[16]
Ruiz-Ponte C, Cilia V, Lambert C, Nicolas JL (1998) Roseobacter gallaeciensis sp. nov., a new marine bacterium isolated from rearings and collectors of the scallop Pecten maximus. Int J Syst Bacteriol 48 Pt 2: 537–542. doi: 10.1099/00207713-48-2-537
[17]
Yoon JH, Kang SJ, Lee SY, Oh TK (2007) Phaeobacter daeponensis sp. nov., isolated from a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 57: 856–861. doi: 10.1099/ijs.0.64779-0
[18]
Zhang DC, Li HR, Xin YH, Liu HC, Chi ZM, et al. (2008) Phaeobacter arcticus sp. nov., a psychrophilic bacterium isolated from the Arctic. Int J Syst Evol Microbiol 58: 1384–1387. doi: 10.1099/ijs.0.65708-0
[19]
Druppel K, Hensler M, Trautwein K, Kossmehl S, Wohlbrand L, et al. (2014) Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade). Environ Microbiol. 16: 218–38. doi: 10.1111/1462-2920.12276
[20]
Kossmehl S, Wohlbrand L, Druppel K, Feenders C, Blasius B, et al. (2013) Subcellular protein localization (cell envelope) in Phaeobacter inhibens DSM 17395. Proteomics 13: 2743–2760. doi: 10.1002/pmic.201300112
[21]
Zech H, Hensler M, Kossmehl S, Druppel K, Wohlbrand L, et al. (2013) Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395. Proteomics 13: 2869–2885. doi: 10.1002/pmic.201200560
[22]
Zech H, Hensler M, Kossmehl S, Druppel K, Wohlbrand L, et al. (2013) Adaptation of Phaeobacter inhibens DSM 17395 to growth with complex nutrients. Proteomics 13: 2851–2868. doi: 10.1002/pmic.201200513
[23]
Berger M, Neumann A, Schulz S, Simon M, Brinkhoff T (2011) Tropodithietic acid production in Phaeobacter gallaeciensis is regulated by N-acyl homoserine lactone-mediated quorum sensing. J Bacteriol 193: 6576–6585. doi: 10.1128/jb.05818-11
[24]
Brinkhoff T, Giebel HA, Simon M (2008) Diversity, ecology, and genomics of the Roseobacter clade: a short overview. Arch Microbiol 189: 531–539. doi: 10.1007/s00203-008-0353-y
[25]
Seyedsayamdost MR, Carr G, Kolter R, Clardy J (2011) Roseobacticides: small molecule modulators of an algal-bacterial symbiosis. J Am Chem Soc 133: 18343–18349. doi: 10.1021/ja207172s
[26]
Seyedsayamdost MR, Case RJ, Kolter R, Clardy J (2011) The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis. Nat Chem 3: 331–335. doi: 10.1038/nchem.1002
[27]
Buddruhs N, Pradella S, Goker M, Pauker O, Pukall R, et al. (2013) Molecular and phenotypic analyses reveal the non-identity of the Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395. Int J Syst Evol Microbiol 63: 4340–4349. doi: 10.1099/ijs.0.053900-0
[28]
Dedieu A, Gaillard JC, Pourcher T, Darrouzet E, Armengaud J (2011) Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J Biol Chem 286: 259–269. doi: 10.1074/jbc.m110.159483
[29]
de Groot A, Dulermo R, Ortet P, Blanchard L, Guerin P, et al. (2009) Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti. PLoS Genet 5: e1000434. doi: 10.1371/journal.pgen.1000434
[30]
Dupierris V, Masselon C, Court M, Kieffer-Jaquinod S, Bruley C (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25: 1980–1981. doi: 10.1093/bioinformatics/btp301
[31]
Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76: 4193–4201. doi: 10.1021/ac0498563
[32]
Zivanovic Y, Armengaud J, Lagorce A, Leplat C, Guerin P, et al. (2009) Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea. Genome Biol 10: R70. doi: 10.1186/gb-2009-10-6-r70
[33]
Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, et al. (2006) Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci U S A 103: 18928–18933. doi: 10.1073/pnas.0606379103
[34]
Baudet M, Ortet P, Gaillard JC, Fernandez B, Guerin P, et al. (2010) Proteomics-based refinement of Deinococcus deserti genome annotation reveals an unwonted use of non-canonical translation initiation codons. Mol Cell Proteomics 9: 415–426. doi: 10.1074/mcp.m900359-mcp200
[35]
Bland C, Bellanger L, Armengaud J (2014) Magnetic Immunoaffinity Enrichment for Selective Capture and MS/MS Analysis of N-Terminal-TMPP-Labeled Peptides. J Proteome Res. in press.
[36]
Preston GM, Studholme DJ, Caldelari I (2005) Profiling the secretomes of plant pathogenic Proteobacteria. FEMS Microbiol Rev 29: 331–360. doi: 10.1016/j.femsre.2004.12.004
[37]
Clair G, Lorphelin A, Armengaud J, Duport C (2013) OhrRA functions as a redox-responsive system controlling toxinogenesis in Bacillus cereus. J Proteomics 94: 527–539. doi: 10.1016/j.jprot.2013.10.024
[38]
Linhartova I, Bumba L, Masin J, Basler M, Osicka R, et al. (2010) RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol Rev 34: 1076–1112. doi: 10.1111/j.1574-6976.2010.00231.x
[39]
Moran MA, Belas R, Schell MA, Gonzalez JM, Sun F, et al. (2007) Ecological genomics of marine Roseobacters. Appl Environ Microbiol 73: 4559–4569. doi: 10.1128/aem.02580-06
[40]
Satchell KJ (2011) Structure and function of MARTX toxins and other large repetitive RTX proteins. Annu Rev Microbiol 65: 71–90. doi: 10.1146/annurev-micro-090110-102943
[41]
Sheahan KL, Cordero CL, Satchell KJ (2007) Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain. EMBO J 26: 2552–2561. doi: 10.1038/sj.emboj.7601700
[42]
Vigil PD, Alteri CJ, Mobley HL (2011) Identification of in vivo-induced antigens including an RTX family exoprotein required for uropathogenic Escherichia coli virulence. Infect Immun 79: 2335–2344. doi: 10.1128/iai.00110-11
