BMC Genomics | |
Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors | |
David J Norman4  Marjorie Chow1  Arianna Mangravita-Novo2  Ute CM Achenbach3  Ana M Bocsanczy4  | |
[1] ICBR Proteomics Core, University of Florida, Gainesville, FL 32610, USA;Burnham Institute for Medical Research at Lake Nona, 6400 Sanger Road, Orlando, FL 32827, USA;Development Lead North-East Europe, Syngenta Agro GmbH, Am Technologiepark 1-5 63477, Maintal, Germany;Department of Plant Pathology, University of Florida, IFAS, Mid-Florida Research and Education Center, 2725 Binion Rd., Apopka, FL 32703, USA | |
关键词: Stress response; Type VI secretion system; Virulent strains at low temperature; Temperature; Bacterial wilt; | |
Others : 1217476 DOI : 10.1186/1471-2164-15-280 |
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received in 2013-11-06, accepted in 2014-04-09, 发布年份 2014 | |
【 摘 要 】
Background
Ralstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).
Results
Four hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.
Conclusions
The putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.
【 授权许可】
2014 Bocsanczy et al.; licensee BioMed Central Ltd.
【 预 览 】
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Figure 1. | 86KB | Image | download |
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【 参考文献 】
- [1]Hurme R, Rhen M: Temperature sensing in bacterial gene regulation - what it all boils down to. Mol Microbiol 1998, 30(1):1-6.
- [2]Ramos JL, Gallegos MT, Marques S, Ramos-Gonzalez MI, Espinosa-Urgel M, Segura A: Responses of gram-negative bacteria to certain environmental stressors. Curr Opin Microbiol 2001, 4(2):166-171.
- [3]Konkel ME, Tilly K: Temperature-regulated expression of bacterial virulence genes. Microbes Infect 2000, 2(2):157-166.
- [4]Rejasse A, Gilois N, Barbosa I, Huillet E, Bevilacqua C, Tran S, Ramarao N, Arnesen LPS, Sanchis V: Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4. Appl Environ Microbiol 2012, 78(8):2553-2561.
- [5]Kimes NE, Grim CJ, Johnson WR, Hasan NA, Tall BD, Kothary MH, Kiss H, Munk AC, Tapia R, Green L, Detter C, Bruce DC, Brettin TS, Colwell RR, Morris PJ: Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus. Isme J 2012, 6(4):835-846.
- [6]Maurelli AT, Sansonetti PJ: Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. P Natl Acad Sci USA 1988, 85(8):2820-2824.
- [7]Elphinstone JG, Allen C, Prior P: The current Bacterial Wilt situation: A global overview. In Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex USA. Edited by Hayward AC. St. Paul, Minnesota: APS Press; 2005:9-28.
- [8]Buddenhagen I, Sequeira L, Kelman A: Designation of races in Pseudomonas solanacearum. Phytopathol 1962, 52(8):726-1962.
- [9]Hayward AC: Characteristics of Pseudomonas solanacearum. J Appl Microbiol 1964, 27(2):265-277.
- [10]Fegan M, Prior P: How complex is the “Ralstonia solanacearum species complex”. In Bacterial Wilt Disease and the Ralstonia Solanacearum Species Complex. Edited by Allen PP C, Hayward AC. Madison, WI: APS Press; 2005:449-462.
- [11]Thurston HD: Bacterial wilt of potatoes in Colombia. Amer Potato J 1963, 40(11):381-390.
- [12]Janse JD, van den Beld HE, Elphinstone J, Simpkins S, Tjou-Tam-Sin NNA, van Vaerenbergh J: Introduction to Europe of Ralstonia solanacearum biovar 2, race 3 in Pelargonium zonale cuttings. J Plant Pathol 2004, 86(2):147-155.
- [13]Ciampi L, Sequeira L: Influence of temperature on virulence of race 3 strains of Pseudomonas solanacearum. Am Potato J 1980, 57(7):307-317.
- [14]Lambert CD: Agricultural bioterrorism protection act of 2002: possession, use, and transfer of biological; agents and toxins; interim and final rule. (7 CFR Part 331). Fed Regist 2002, 67:76908-76938.
- [15]Duan Y, Norman D, Gabriel D: Distribution and sequence analysis of putative determinants of race, biovar and cold tolerance factors of Ralstonia solanacearum. Phytopathol 2005, 95(6):S26.
- [16]Denny TP, Milling AS, Bhakta VG, Allen C: Ralstonia solanacearum race 3 biovar 2 strains are not uniquely cold tolerant in vitro. Phytopathol 2007, 97(7):S28.
- [17]Bocsanczy AM, Achenbach UC, Mangravita-Novo A, Yuen JM, Norman DJ: Comparative effect of low temperature on virulence and twitching motility of Ralstonia solanacearum strains present in Florida. Phytopathol 2012, 102(2):185-194.
- [18]Beranova-Giorgianni S: Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations. Trac-Trend Anal Chem 2003, 22(5):273.
- [19]Giglio MG, Collmer CW, Lomax J, Ireland A: Special issue: gene ontology for microbiologists applying the gene ontology in microbial annotation. Trends Microbiol 2009, 17(7):262-268.
- [20]Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S: AmiGO: online access to ontology and annotation data. Bioinformatics 2009, 25(2):288-289.
- [21]Flores-Cruz Z, Allen C: Ralstonia solanacearum encounters an oxidative environment during tomato infection. Mol Plant Microbe In 2009, 22(7):773-782.
- [22]Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ: Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 1998, 180(8):1979-1987.
- [23]Schlegel HG, Vonbartheld R, Gottschalk G: Formation and utilization of poly-beta-hydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature 1961, 191(478):463.
- [24]Handrick R, Reinhardt S, Kimmig P, Jendrossek D: The “intracellular” poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. J Bacteriol 2004, 186(21):7243-7253.
- [25]Hennequin C, Collignon A, Karjalainen T: Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress. Microb Pathogenesis 2001, 31(5):255-260.
- [26]Thomas JG, Baneyx F: ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. Mol Microbiol 2000, 36(6):1360-1370.
- [27]Hernandez-Romero D, Solano F, Sanchez-Amat A: Polyphenol oxidase activity expression in Ralstonia solanacearum. Appl Environ Microbiol 2005, 71(11):6808-6815.
- [28]Colburn-Clifford JM, Scherf JM, Allen C: Ralstonia solanacearum Dps contributes to oxidative stress tolerance and to colonization of and virulence on tomato plants. Appl Environ Microbiol 2010, 76(22):7392-7399.
- [29]Xu XQ, Pan SQ: An agrobacterium catalase is a virulence factor involved in tumorigenesis. Mol Microbiol 2000, 35(2):407-414.
- [30]Liu H, Kang Y, Genin S, Schell MA, Denny TP: Twitching motility of Ralstonia solanacearum requires a type IV pilus system. Microbiol (Reading) 2001, 147(12):3215-3229.
- [31]Kang Y, Liu H, Genin S, Schell MA, Denny TP: Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence. Mol Microbiol 2002, 46(2):427-437.
- [32]Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S, Waltz T, Joachimiak A, Mekalanos JJ: A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006, 312(5779):1526-1530.
- [33]Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ: Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. P Natl Acad Sci USA 2006, 103(5):1528-1533.
- [34]Sarris PF, Skandalis N, Kokkinidis M, Panopoulos NJ: In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars. Mol Plant Pathol 2010, 11(6):795-804.
- [35]Zheng J, Leung KY: Dissection of a type VI secretion system in Edwardsiella tarda. Mol Microbiol 2007, 66(5):1192-1206.
- [36]Haapalainen M, Mosorin H, Dorati F, Wu RF, Roine E, Taira S, Nissinen R, Mattinen L, Jackson R, Pirhonen M, Lin NC: Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for fitness for competition against bacteria and yeasts. J Bacteriol 2012, 194(18):4810-4822.
- [37]Wu HY, Chung PC, Shih HW, Wen SR, Lai EM: Secretome analysis uncovers an hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. J Bacteriol 2008, 190(8):2841-2850.
- [38]Bingle LEH, Bailey CM, Pallen MJ: Type VI secretion: a beginner’s guide. Curr Opin Microbiol 2008, 11(1):3-8.
- [39]Bladergroen MR, Badelt K, Spaink HP: Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe In 2003, 16(1):53-64.
- [40]da Silva FG, Shen YW, Dardick C, Burdman S, Yadav RC, de Leon AL, Ronald PC: Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response. Mol Plant Microbe In 2004, 17(6):593-601.
- [41]Lomovskaya O, Lewis K: Emr, an Escherichia coli locus for multidrug resistance. P Natl Acad Sci USA 1992, 89(19):8938-8942.
- [42]Colmer JA, Fralick JA, Hamood AN: Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae. Mol Microbiol 1998, 27(1):63-72.
- [43]Xu J, Zheng HJ, Liu L, Pan ZC, Prior P, Tang B, Xu JS, Zhang H, Tian Q, Zhang LQ, Feng J: Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82. J Bacteriol 2011, 193(16):4261-4262.
- [44]Stevens P, van Overbeek LS, van Elsas JD: Ralstonia solanacearum Delta PGI-1 strain KZR-5 is affected in growth, response to cold stress and invasion of tomato. Microb Ecol 2011, 61(1):101-112.
- [45]Liu HL, Zhang SP, Schell MA, Denny TP: Pyramiding, unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Mol Plant Microbe In 2005, 18(12):1296-1305.
- [46]Boucher CA, Gough CL, Arlat M: Molecular genetics of pathogenicity determinants of Pseudomonas solanacearum with special emphasis on hrp genes. Annu Rev Phytopathol 1992, 30:443-461.
- [47]Denny TP, Baek SR: Genetic evidence that extracellular polysaccharide is a virulence factor of Pseudomonas solanacearum. Mol Plant Microbe In 1991, 4(2):198-206.
- [48]Brencic A, Winans SC: Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiol Mol Biol R 2005, 69(1):155.
- [49]Yao J, Allen C: Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum. J Bacteriol 2006, 188(10):3697-3708.
- [50]Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ: Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. P Natl Acad Sci USA 2007, 104(39):15508-15513.
- [51]Schell MA: Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network. Annu Rev Phytopathol 2000, 38:263-292.
- [52]Brumbley SM, Carney BF, Denny TP: Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J Bacteriol 1993, 175(17):5477-5487.
- [53]Huang JZ, Carney BF, Denny TP, Weissinger AK, Schell MA: A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum. J Bacteriol 1995, 177(5):1259-1267.
- [54]Nissinen RM, Ytterberg AJ, Bogdanove AJ KJVANW, Beer SV: Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels. Mol Plant Pathol 2007, 8(1):55-67.
- [55]Keller A, Nesvizhskii AI, Kolker E, Aebersold R: Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 2002, 74(20):5383-5392.
- [56]Nesvizhskii AI, Keller A, Kolker E, Aebersold R: A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 2003, 75(17):4646-4658.
- [57]Jahn CE, Charkowski AO, Willis DK: Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. J Microbiol Meth 2008, 75(2):318-324.