The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Altered Gene Expression and Intracellular Changes of the Viable But Nonculturable State in Ralstonia solanacearum by Copper Treatment

  • Received : 2013.07.09
  • Accepted : 2013.08.21
  • Published : 2013.12.01
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Abstract

Environmental stresses induce several plant pathogenic bacteria into a viable but nonculturable (VBNC) state, but the basis for VBNC is largely uncharacterized. We investigated the physiology and morphology of the copper-induced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of $200{\mu}M$ copper sulfate to the liquid microcosm completely suppressed bacterial colony formation on culture media; however, LIVE/DEAD BacLight bacterial viability staining showed that the bacterial cells maintained viability, and that the viable cells contain higher level of DNA. Based on electron microscopic observations, the bacterial cells in the VBNC state were unchanged in size, but heavily aggregated and surrounded by an unknown extracellular material. Cellular ribosome contents, however, were less, resulting in a reduction of the total RNA in VBNC cells. Proteome comparison and reverse transcription PCR analysis showed that the Dps protein production was up-regulated at the transcriptional level and that 2 catalases/peroxidases were present at lower level in VBNC cells. Cell aggregation and elevated levels of Dps protein are typical oxidative stress responses. $H_2O_2$ levels also increased in VBNC cells, which could result if catalase/peroxidase levels are reduced. Some of phenotypic changes in VBNC cells of R. solanacearum could be an oxidative stress response due to $H_2O_2$ accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.

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References

  1. Alexander, E., Pham, D. and Steck, T. R. 1999. The viable-butnonculturable conditions is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl. Environ. Microbiol. 65:3754-3756.
  2. Almiron, M., Link, A., Furlong, D. and Kolter, R. 1992. A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev. 6:2646-2654. https://doi.org/10.1101/gad.6.12b.2646
  3. Alvarez, B., Lopez, M. M. and Biosca, E. G. 2008. Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms. Microbiology 154:3590-3598. https://doi.org/10.1099/mic.0.2008/019448-0
  4. Blat, Y. and Eisenbach, M. 1995. Tar-dependent and -independent pattern formation by Salmonella typhimurium. J. Bacteriol. 177:1683-1691. https://doi.org/10.1128/jb.177.7.1683-1691.1995
  5. Boucher, C. A., Barberis, P., Trigalet, A. P. and Demery, D. A. 1985. Transposon mutagenesis of Pseudomonas solanacearum: isolation of Tn5-induced avirulent mutants. J. Gen. Microbiol. 131:2449-2457.
  6. Boulos, L., Prevost, M., Barbeau, B., Coallier, J. and Desjardins, R. 1999. $LIVE/DEAD^{(R)}$ BacLightTM: application of a new rapid staining method for direct enumeration of viable ad total bacteria in drinking water. J. Microbiol. Meth. 37:77-86. https://doi.org/10.1016/S0167-7012(99)00048-2
  7. Brumbley, S. M. and T. P. Denny. 1990. Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence. J. Bacteriol. 172:5677-5685. https://doi.org/10.1128/jb.172.10.5677-5685.1990
  8. Budrene, E. O. and Berg, H. C. 1991. Complex patterns formed by motile cells of Escherichia coli. Nature 349:630-633. https://doi.org/10.1038/349630a0
  9. Caruso, P., Palomo, J. L., Bertolini, E., Alvarez, B., Lopez, M. M. and Biosca, E. G. 2005. Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river; recovery of stressed cells at low temperature. Appl. Environ. Microbiol. 71:140-148. https://doi.org/10.1128/AEM.71.1.140-148.2005
  10. Colburn-Clifford, J. M., Scherf, J. M. and Allen, C. 2010. Ralstonia solanacearum Dps contributes to oxidative stress tolerance and to colonization of and virulence on tomato plants. Appl. Environ. Microbiol. 76:7392-7399. https://doi.org/10.1128/AEM.01742-10
  11. Coutinho, T. A. 2005. Introduction and prospectus on the survival of R. solanacearum, p. 29-38. In C. Allen, P. Prior and A. C. Hayward, eds., Bacterial wilt disease and the Ralstonia solanacearum species complex. APS press, St. Paul, MN.
  12. Ghezzi, J. I. and Steck, J. M. 1999. Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil. FEMS Microbiol. Ecol. 30:203-208. https://doi.org/10.1111/j.1574-6941.1999.tb00648.x
  13. Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R. and Hogle, J. M. 1998. The crystal structure of Dps, a ferritin homolog that binds and protects DNA. Nature Struct. Biol. 5:294-303. https://doi.org/10.1038/nsb0498-294
  14. Grey, B. and Steck, T. R. 2001. The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl. Environ. Microbiol. 67:3866-3872. https://doi.org/10.1128/AEM.67.9.3866-3872.2001
  15. Hayward, A. C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29:65-87. https://doi.org/10.1146/annurev.py.29.090191.000433
  16. Holmgren, G. G. S., Meyer, M., Chaney, R. and Daniels, R. 1993. Cadmium, lead, zinc, copper, and nickel in agricultural soils of the United States of America. J. Environ. Qual. 22:335-348.
  17. Imazaki, I. and Nakaho, K. 2009. Temperature-upshift-mediated revival from the sodium-pyruvate-recoverable viable but nonculturable state induced by low temperature in Ralstonia solanacearum: linear regression analysis. J. Gen. Plant Pathol. 75:213-226. https://doi.org/10.1007/s10327-009-0166-0
  18. Jeong, Y., Kim, J., Kang, Y., Lee, S. and Hwang, I. 2007. Genetic diversity and distribution of Korean isolates of Ralstonia solanacearum. Plant Dis. 91:1277-1287. https://doi.org/10.1094/PDIS-91-10-1277
  19. Kell, D., Kaprelyants, A., Weichart, D., Harwood, C. and Barer, M. 1998. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Leeuwenhoek. 73:169-187. https://doi.org/10.1023/A:1000664013047
  20. Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693-695.
  21. Kelman, A. 1956. Survival of Pseudomonas solanacearum in water. Phytopathology 46:16-17.
  22. Kim, S. T., Kim, S. G., Kang, Y. H., Wang, Y., Kim, J.-Y., Yi, N., Kim, J.-K., Rakwal, R., Koh, J.-K. and Kang, K. Y. 2008. Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death. J. Proteome Res. 7:1750-1760. https://doi.org/10.1021/pr700878t
  23. Kim, Y. H., Kim, K. S. and Riggs, R. D. 2012. Initial subcellular responses of susceptible and resistant soybeans infected with the soybean cyst nematode. Plant Pathol. J. 28:401-408. https://doi.org/10.5423/PPJ.OA.04.2012.0054
  24. Kogure, K., Simidu, U. and Taga, N. 1979. A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25:415-420. https://doi.org/10.1139/m79-063
  25. Kong, I.-S., Bates, T. C., Hulsmann, A., Hassan, H. and Oliver, J. D. 2004. Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiol. Ecol. 50:133-142. https://doi.org/10.1016/j.femsec.2004.06.004
  26. Lahtinen, S. J., Ahokoski, H., Reinikainen, J. P., Gueimonde, M., Nurmi, J., Ouwehand, A. C. and Salminen, S. J. 2008. Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria. Lett. Appl. Microbiol. 46:693-698. https://doi.org/10.1111/j.1472-765X.2008.02374.x
  27. Lee, Y. H., Choi, C. W., Kim, S. H., Yun, J. G., Chang, S. W., Kim, Y. S. and Hong, J. K. 2012. Chemical pesticides and plant essential oils for disease control of tomato bacterial wilt. Plant Pathol. J. 28:32-39. https://doi.org/10.5423/PPJ.OA.10.2011.0200
  28. Loreto, F. and Velikova, V. 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quences ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol. 127:1781-1787. https://doi.org/10.1104/pp.010497
  29. Martinez, A. and Kolter, R. 1997. Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps. J. Bacteriol. 179:5188-5194. https://doi.org/10.1128/jb.179.16.5188-5194.1997
  30. Mergaert, P., Uchiumi, T., Alunni, B., Evanno, G., Cheron, A., Catrice, O., Mausset, A. E., Barloy-Hubler, F., Galibert, F., Kondorosi, A. and Kondorosi, E. 2006. Eukaryotic control of bacterial cell cycle and differentiation in the Rhizobiumlegume symbiosis. Proc. Natl. Acad. Sci. USA 103:5230-5235. https://doi.org/10.1073/pnas.0600912103
  31. Michelsen, O., Hansen, F. G., Albrechtsen, B. and Jensen, P. R. 2010. The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid. J. Bacteriol. 192:1058-1065. https://doi.org/10.1128/JB.00900-09
  32. Mizunoe, Y., Wai, S. N., Takade, A. and Yoshida, S. 1999. Restoration of culturability of starvation-stressed Escherichia coli O157 cells by using $H_2O_2$-degrading compounds. Arch. Microbiol. 172:63-67. https://doi.org/10.1007/s002030050741
  33. Oliver, J. D. 2010. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34:415-425. https://doi.org/10.1111/j.1574-6976.2009.00200.x
  34. Ordax, M., Marco-Noales, E., Lopez, M. M. and Biosca, E. G. 2006. Survival strategy of Erwinia amylovora against copper:induction of the viable-but-nonculturable state. Appl. Environ. Microbiol. 72:3482-3488. https://doi.org/10.1128/AEM.72.5.3482-3488.2006
  35. Potter, M., Muller, H., Reinecke, F., Wieczorek, R., Fricke, F., Bowien, B., Friedrich, B. and Steinbuchel, A. 2004. The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology 150:2301-2311. https://doi.org/10.1099/mic.0.26970-0
  36. Rodriguez, G. G., Phipps, D., Ishiguro, K. and Ridgway, H. F. 1992. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58:1801-1808.
  37. Saddler, G. S. 2005. Management of bacterial wilt disease, p.121-132. In C. Allen, P. Prior and A. C. Hayward, eds., Bacterial wilt disease and the Ralstonia solanacearum species complex. APS press, St. Paul, MN.
  38. Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Arlat, M., Billault, A., Brottier, P., Camus, J. C., Cattolico, L., Chandler, M., Choisne, N., Claudel-Renard, C., Cunnac, S., Demange, N., Gaspin, C., Lavie, M., Moisan, A., Robert, C., Saurin, W., Schiex, T., Siguier, P., Thebault, P., Whalen, M., Wincker, P., Levy, M., Weissenbach, J. and Boucher, C. A. 2002. Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497-502. https://doi.org/10.1038/415497a
  39. Saumaa, S., Tover, A., Tark, M., Tegova, R. and Kivisaar, M. 2007. Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida. J. Bacteriol. 189:5504-5514. https://doi.org/10.1128/JB.00518-07
  40. Schaad, N. W., Jones, J. B. and Chun, W. 2001. Laboratory guide for identification of plant pathogenic bacteria, 3rd ed., APS press, St. Paul, MN.
  41. Shapiro, J. A. 1988. Bacteria as multicellular organisms. Sci. Am. 258:62-69.
  42. Tobiason, D. M. and Seifert, H. S. 2010. Genomic content of Neisseria species. J. Bacteriol. 192:2160-2168. https://doi.org/10.1128/JB.01593-09
  43. van Elsas, J. D., Kastelein, P., de Vries, P. M. and van Overbeek, L. S. 2001. Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water. Can. J. Microbiol. 47:842-854. https://doi.org/10.1139/cjm-47-9-842
  44. van Elsas, J. D., Kastelein, P., van Bekkum, P., van der Wolf, J. M., de Vries, P. M. and van Overbeek, L. S. 2000. Survival of Ralstonia solanacearum biovar 2, the causative agent of potato brown rot, in field and microcosm soils in temperate climates. Phytopathology 90:1358-1366. https://doi.org/10.1094/PHYTO.2000.90.12.1358
  45. Xu, H., Roberts, N., Singleton, F. L., Attwell, R. W., Grimes, D. J. and Colwell, R. R. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholera in the estuarine and marine environment. Microb. Ecol. 8:313-323. https://doi.org/10.1007/BF02010671
  46. Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. and Nishiuchi, Y. Y. 1995. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: Proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis 1969) comb. nov. Microbiol. Immunol. 39:897-904. https://doi.org/10.1111/j.1348-0421.1995.tb03275.x

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