Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
권호 표지
DOI QR코드

DOI QR Code

Sigma S Involved in Bacterial Survival of Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum 생존에 관여하는 Sigma S 역할

  • Hye Kyung Choi (Department of Applied Bioscience, Dong-A University) ;
  • Eun Jeong Jo (Department of Applied Bioscience, Dong-A University) ;
  • Jee Eun Heo (Department of Applied Bioscience, Dong-A University) ;
  • Hyun Gi Kong (Department of Plant Medicine, Chungbuk National University) ;
  • Seon-Woo Lee (Department of Applied Bioscience, Dong-A University)
  • 최혜경 (동아대학교 응용생명과학과) ;
  • 조은정 (동아대학교 응용생명과학과) ;
  • 허지은 (동아대학교 응용생명과학과) ;
  • 공현기 (충북대학교 식물의학과) ;
  • 이선우 (동아대학교 응용생명과학과)
  • Received : 2024.05.06
  • Accepted : 2024.05.21
  • Published : 2024.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Ralstonia pseudosolanacearum, a plant pathogenic bacterium that can survive for a long time in soil and water, causes lethal wilt in the Solanaceae family. Sigma S is a part of the RNA polymerase complex, which regulates gene expression during bacterial stress response or stationary phase. In this study, we investigated the role of sigma S in R. pseudosolanacearum under stress conditions using a rpoS-defective mutant strain of R. pseudosolanacearum and its wild-type strain. The phenotypes of rpoS-defective mutant were complemented by introducing the original rpoS gene. There were no differences observed in bacterial growth rate and exopolysaccharide production between the wild-type strain and the rpoS mutant. However, the wild-type strain responded more sensitively to nutrient deficiency compared to the mutant strain. Under the nutrient deficiency, the rpoS mutant maintained a high bacterial viability for a longer period, while the viability of the wild-type strain declined rapidly. Furthermore, a significant difference in pH was observed between the culture supernatant of the wild-type strain and the mutant strain. The pH of the culture supernatant for the wild-type strain decreased rapidly during bacterial growth, leading to medium acidification. The rapid decline in the wild-type strain's viability may be associated with medium acidification and bacterial sensitivity to acidity during transition to the stationary phase. Interestingly, the rpoS mutant strain cannot utilize acetic acid, D-alanine, D-trehalose, and L-histidine. These results suggest that sigma S of R. pseudosolanacearum regulates the production or utilization of organic acids and controls cell death during stationary phase under nutrient deficiency.

Ralstonia pseudosolanacearum은 토양과 물에서 오랫동안 생존하고, 가지과 작물에 심각한 풋마름병을 일으키는 식물병원세균이다. Simga S는 세균의 스트레스 환경에서 반응 또는 정지기 동안 유전자 발현을 조절하는 RNA 중합효소 복합체의 일부인 단백질이다. 본 연구는 스트레스 조건에서 R.pseudosolanacearum의 sigma S의 역할을 조사하기 위해서, R.pseudosolanacearum의 GMI1000 균주의 sigma S를 암호화하는 rpoS 유전자 변이체를 준비하여 야생형 균주와 세균의 특징을 비교하였다. 아울러 rpoS 유전자 역할은 원래 유전자를 변이체에 도입하여 rpoS 유전자 표현형 회복을 확인하였다. 야생형 균주와 rpoS 결여 변이체는 생장 속도, 외피다당류 생산, 식물체에서 병원성, 식물 세포벽 분해 효소 활성에서 차이를 보이지 않았다. 그러나 야생형 균주는 영양분결핍 조건에서 변이체보다 더 민감하게 반응하였고 과산화수소가 첨가된 조건에서 변이체보다 덜 민감하게 반응하였다. 흥미롭게도 영양분결핍 조건에서 rpoS 결여 변이체에서는 장기간 생균수를 유지하지만, 같은 조건에서 야생형 균주 생균수는 빠르게 감소하였다. 그리고 두 균주 배양액 pH를 측정한 결과, 야생형 균주와 변이체 간에 상당한 차이가 나타났다. 야생형 균주는 생장하면서 빠르게 배지의 pH가 감소하여 산성화되었다. 그러므로 야생형 균주의 빠른 사멸은 배지가 산성화되면서 정지기 상태 세균의 산성 pH에 대한 민감도 때문일 것이다. Biolog 분석으로 rpoS 변이체는 acetic acid, D-alanine, D-trehalose, L-histidine을 이용하지 못함을 확인하였다. 본 연구 결과는 R. pseudosolanacearum 세균의 sigma S가 영양분결핍 조건에서 정지기 동안 유기산 생산 또는 이용을 조절하며 정지기 세포사멸도 조절하는 것을 보여준다.

Keywords

Acknowledgement

This research was supported by the National Research Foundation of Korea (NRF) grant (No. 2020R1A2C3005453 and 2020R1A6A1A03047729), Biomaterials Specialized Graduate Program funded by the Korea government (MSIT, MOE, ME), Republic of Korea.

References

  1. Arlat, M., Gough, C. L., Zischek, C., Barberis, P. A., Trigalet, A. and Boucher, C. A. 1992. Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum. Mol. Plant Microbe Interact. 5: 187-193.
  2. Astaurova, O. B., Bass, I. A. and Khmel', I. A. 2007. Suggested interrelationships of RNA-polymerase sigma S subunit and nitrogen control system in Pseudomonas chlororaphis. Genetika 43: 1026-1031. (In Russian)
  3. Badger, J. L. and Miller, V. L. 1995. Role of RpoS in survival of Yersinia enterocolitica to a variety of environmental stresses. J. Bacteriol. 177: 5370-5373.
  4. Baker, C. J. and Orlandi, E. W. 1995. Active oxygen in plant pathogenesis. Annu. Rev. Phytopathol. 33: 299-321.
  5. Bergsma-Vlami, M., van de Bilt, J. L. J., Tjou-Tam-Sin, N. N. A., Westenberg, M., Meekes, E. T. M., Teunissen, H. A. S. et al. 2018. Phylogenetic assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) isolated from Rosa spp. Plant Dis. 102: 2258-2267.
  6. Boucher, C. A., Barberis, P. A., 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.
  7. Boyer, H. W. and Roulland-Dussoix, D. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41: 459-465.
  8. Fang, F. C., Libby, S. J., Buchmeier, N. A., Loewen, P. C., Switala, J., Harwood, J. et al. 1992. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc. Natl. Acad. Sci. U S A 89: 11978-11982.
  9. Figurski, D. H. and Helinski, D. R. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. U S A 76: 1648-1652.
  10. Flavier, A. B., Schell, M. A. and Denny, T. P. 1998. An RpoS (sigmaS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum. Mol. Microbiol. 28: 475-486.
  11. Genin, S. and Boucher, C. 2002. Ralstonia solanacearum: secrets of a major pathogen unveiled by analysis of its genome. Mol. Plant Pathol. 3: 111-118.
  12. Guillemet, M. L. and Moreau, P. L. 2008. Fur-dependent detoxification of organic acids by rpoS mutants during prolonged incubation under aerobic, phosphate starvation conditions. J. Bacteriol. 190: 5567-5575.
  13. Hayward, A. C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29: 65-87.
  14. Hayward, A. C. 1994. Systematics and phylogeny of Pseudomonas solanacearum and related bacteria. In: Bacterial Wilt: the Disease and its Causative Agent Pseudomonas solanacearum, eds. by A. C. Haywar and G. L. Hartman, pp. 127-135. CAB International, Oxford, UK.
  15. Hayward, A. C. 2000. Ralstonia solanacearum. In: Encyclopedia of Microbiology Vol. 4, ed. by J. Lederberg, pp. 32-42. Academic Press, San Diego, CA, USA.
  16. Hengge-Aronis, R. 1996a. Regulation of gene expression during entry into stationary phase. In: Escherichia coli and Salmonella: Cellular and Molecular Biology, eds. by F. C. Neidhardt, R. I. Curtis, J. L. Ingram, E. C. C. Lin, K. B. Low, B. Magasanik, et al., pp. 1497-1512. American Society for Microbiology Press, Washington, DC, USA.
  17. Hengge-Aronis, R. 1996b. Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli. Mol. Microbiol. 21: 887-893.
  18. Hikichi, Y., Yoshimochi, T., Tsujimoto, S., Shinohara, R., Nakaho, K., Kanda, A. et al. 2007. Global regulation of pathogenicity mechanism of Ralstonia solanacearum. Plant Biotechnol. 24: 149-154.
  19. Hwang, S. Y. 2006. Alanine racemase specific activity detection and serine racemase expression and specific activity detection. M. S. thesis. Inha University, Incheon, Korea. 1-69 pp.
  20. Ito, S., Ushuima, Y., Fujii, T., Tanaka, S., Kameya-Iwaki, M., Yoshiwara, S. et al. 1998. Detection of viable cells of Ralstonia solanacearum in soil using a semiselective medium and a PCR technique. J. Phytopathol. 146: 379-384.
  21. Keen, N. T., Tamaki, S., Kobayashi, D. and Trollinger, D. 1988. Improved broad-host-range plasmids for DNA cloning in gramnegative bacteria. Gene 70: 191-197.
  22. Kelman, A. 1954. The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. Phytopathology 44: 693-695.
  23. Kolter, R., Siegele, D. A. and Tormo, A. 1993. The stationary phase of the bacterial life cycle. Annu. Rev. Microbiol. 47: 855-874.
  24. Lange, R. and Hengge-Aronis, R. 1991. Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S. J. Bacteriol. 173: 4474-4481.
  25. Loewen, P. C. and Hengge-Aronis, R. 1994. The role of the sigma factor sigma S (KatF) in bacterial global regulation. Annu. Rev. Microbiol. 48: 53-80.
  26. McCann, M. P., Kidwell, J. P. and Matin, A. 1991. The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J. Bacteriol. 173: 4188-4194.
  27. Miller, J. H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, New York, NY, USA.
  28. Murillo, J., Shen, H., Gerhold, D., Sharma, A., Cooksey, D. A. and Keen, N. T. 1994. Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23. Plasmid 31: 275-287.
  29. Roberts, D. P., Denny, T. P. and Schell, M. A. 1988. Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity. J. Bacteriol. 170: 1445-1451.
  30. Safni, I., Cleenwerck, I., De Vos, P., Fegan, M., Sly, L. and Kappler, U. 2014. Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov. Int. J. Syst. Evol. Microbiol. 64: 3087-3103.
  31. Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Arlat, M. et al. 2002. Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415: 497-502.
  32. Sambrook, J., Fritsch, E. F. and Maniatis, T. A. 1989. Molecular Cloning: A Laboratory Manual. Vol. 1-3. Cold Spring Harbor, New York, NY, USA.
  33. Sarniguet, A., Kraus, J., Henkels, M. D., Muehlchen, A. M. and Loper, J. E. 1995. The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5. Proc. Natl. Acad. Sci. U S A 92: 12255-12259.
  34. 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, USA.
  35. Song, J. S. and Lee, Y. N. 2006. D-alaninepeptidase increases the vulnerability of bacterial cells to osmotic stress and antibiotics. Kor. J. Microbiol. 42: 299-305. (In Korean)
  36. Wicker, E., Grassart, L., Coranson-Beaudu, R., Mian, D., Guilbaud, C., Fegan, M. et al. 2007. Ralstonia solanacearum strains from martinique (French West Indies) exhibiting a new pathogenic potential. Appl. Environ. Microbiol. 73: 6790-6801.