Skip to main content

Advertisement

SpringerLink
Log in

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog, Rsh Bant

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Battesti A, Bouveret E (2009) Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein–SpoT interaction. J Bacteriol 191(2):616–624. doi:10.1128/jb.01195-08

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Boes N, Schreiber K, Schobert M (2008) SpoT-triggered stringent response controls usp gene expression in Pseudomonas aeruginosa. J Bacteriol 190(21):7189–7199. doi:10.1128/jb.00600-08

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  4. Cashel M (1975) Regulation of bacterial ppGpp and pppGpp. Annu Rev Microbiol 29:301–318. doi:10.1146/annurev.mi.29.100175.001505

    Article  CAS  PubMed  Google Scholar 

  5. Chatterji D, Ojha AK (2001) Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 4(2):160–165

    Article  CAS  PubMed  Google Scholar 

  6. Chen Y, Tenover FC, Koehler TM (2004) Beta-lactamase gene expression in a penicillin-resistant Bacillus anthracis strain. Antimicrob Agents Chemother 48(12):4873–4877. doi:10.1128/aac.48.12.4873-4877.2004

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Chitlaru T, Gat O, Grosfeld H, Inbar I, Gozlan Y, Shafferman A (2007) Identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the Bacillus anthracis secretome. Infect Immun 75(6):2841–2852. doi:10.1128/iai.02029-06

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Chung MC, Popova TG, Millis BA, Mukherjee DV, Zhou W, Liotta LA, Petricoin EF, Chandhoke V, Bailey C, Popov SG (2006) Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors. J Biol Chem 281(42):31408–31418. doi:10.1074/jbc.M605526200

    Article  CAS  PubMed  Google Scholar 

  9. Evans DG, Wardlaw AC (1953) Gelatinase and collagenase production by certain species of Bacillus. J Gen Microbiol 8(3):481–487

    Article  CAS  PubMed  Google Scholar 

  10. Foster TJ, Hook M (1998) Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 6(12):484–488

    Article  CAS  PubMed  Google Scholar 

  11. Fouet A, Mock M (2006) Regulatory networks for virulence and persistence of Bacillus anthracis. Curr Opin Microbiol 9(2):160–166. doi:10.1016/j.mib.2006.02.009

    Article  CAS  PubMed  Google Scholar 

  12. Gohar M, Gilois N, Graveline R, Garreau C, Sanchis V, Lereclus D (2005) A comparative study of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis extracellular proteomes. Proteomics 5(14):3696–3711. doi:10.1002/pmic.200401225

    Article  CAS  PubMed  Google Scholar 

  13. Gong L, Takayama K, Kjelleberg S (2002) Role of spoT-dependent ppGpp accumulation in the survival of light-exposed starved bacteria. Microbiology 148(Pt 2):559–570

    CAS  PubMed  Google Scholar 

  14. Gupta S, Pandit SB, Srinivasan N, Chatterji D (2002) Proteomics analysis of carbon-starved Mycobacterium smegmatis: induction of Dps-like protein. Protein Eng 15(6):503–512

    Article  CAS  PubMed  Google Scholar 

  15. Halsey TA, Vazquez-Torres A, Gravdahl DJ, Fang FC, Libby SJ (2004) The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence. Infect Immun 72(2):1155–1158

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Jung KH, Seo GM, Yoon JW, Park KS, Kim JC, Kim SJ, Oh KG, Lee JH, Chai YG (2008) Protein expression pattern of murine macrophages treated with anthrax lethal toxin. Biochim Biophys Acta 1784(11):1501–1506. doi:10.1016/j.bbapap.2008.06.016

    Article  CAS  PubMed  Google Scholar 

  17. Kim S, Watanabe K, Suzuki H, Watarai M (2005) Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication. Microbiology 151(Pt 5):1607–1617. doi:10.1099/mic.0.27782-0

    Article  CAS  PubMed  Google Scholar 

  18. Kim SK, Shahid S, Kim SH, Park JH, Lee HT, Jung KH, Chai YG (2012) Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature. Lett Appl Microbiol 54(4):306–312. doi:10.1111/j.1472-765X.2012.03209.x

    Article  CAS  PubMed  Google Scholar 

  19. Kvint K, Nachin L, Diez A, Nystrom T (2003) The bacterial universal stress protein: function and regulation. Curr Opin Microbiol 6(2):140–145

    Article  CAS  PubMed  Google Scholar 

  20. Lee VT, Schneewind O (2001) Protein secretion and the pathogenesis of bacterial infections. Genes Dev 15(14):1725–1752. doi:10.1101/gad.896801

    Article  CAS  PubMed  Google Scholar 

  21. Magnusson LU, Farewell A, Nystrom T (2005) ppGpp: a global regulator in Escherichia coli. Trends Microbiol 13(5):236–242. doi:10.1016/j.tim.2005.03.008

    Article  CAS  PubMed  Google Scholar 

  22. Miyoshi S, Shinoda S (2000) Microbial metalloproteases and pathogenesis. Microbes Infect 2(1):91–98 S128645790000280X [pii]

    Article  CAS  PubMed  Google Scholar 

  23. Mock M, Fouet A (2001) Anthrax. Annu Rev Microbiol 55:647–671. doi:10.1146/annurev.micro.55.1.64755/1/647

    Article  CAS  PubMed  Google Scholar 

  24. Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64(3):548–572

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Pajuaba AC, Silva DA, Almeida KC, Cunha-Junior JP, Pirovani CP, Camillo LR, Mineo JR (2012) Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle. Proteomics 12(6):820–831. doi:10.1002/pmic.201100185

    Article  CAS  PubMed  Google Scholar 

  26. Persson O, Valadi A, Nystrom T, Farewell A (2007) Metabolic control of the Escherichia coli universal stress protein response through fructose-6-phosphate. Mol Microbiol 65(4):968–978. doi:10.1111/j.1365-2958.2007.05838.x

    Article  CAS  PubMed  Google Scholar 

  27. Piggot PJ, Hilbert DW (2004) Sporulation of Bacillus subtilis. Curr Opin Microbiol 7(6):579–586. doi:10.1016/j.mib.2004.10.001

    Article  CAS  PubMed  Google Scholar 

  28. Popov SG, Popova TG, Hopkins S, Weinstein RS, MacAfee R, Fryxell KJ, Chandhoke V, Bailey C, Alibek K (2005) Effective antiprotease-antibiotic treatment of experimental anthrax. BMC Infect Dis 5:25. doi:10.1186/1471-2334-5-25

    Article  PubMed Central  PubMed  Google Scholar 

  29. Primm TP, Andersen SJ, Mizrahi V, Avarbock D, Rubin H, Barry CE 3rd (2000) The stringent response of Mycobacterium tuberculosis is required for long-term survival. J Bacteriol 182(17):4889–4898

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Ratnayake-Lecamwasam M, Serror P, Wong KW, Sonenshein AL (2001) Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev 15(9):1093–1103. doi:10.1101/gad.874201

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Reiss S, Pane-Farre J, Fuchs S, Francois P, Liebeke M, Schrenzel J, Lindequist U, Lalk M, Wolz C, Hecker M, Engelmann S (2012) Global analysis of the Staphylococcus aureus response to mupirocin. Antimicrob Agents Chemother 56(2):787–804. doi:10.1128/aac.05363-11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Sonenshein AL (2005) CodY, a global regulator of stationary phase and virulence in Gram-positive bacteria. Curr Opin Microbiol 8(2):203–207. doi:10.1016/j.mib.2005.01.001

    Article  CAS  PubMed  Google Scholar 

  33. Sterne M (1937) Variation in Bacillus anthracis. Onderstepoort J Vet Sci Anim Ind 8:271–349

    CAS  Google Scholar 

  34. Tu WY, Pohl S, Gizynski K, Harwood CR (2012) The iron-binding protein Dps2 confers peroxide stress resistance on Bacillus anthracis. J Bacteriol 194(5):925–931. doi:10.1128/jb.06005-11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Tu WY, Pohl S, Gray J, Robinson NJ, Harwood CR, Waldron KJ (2012) Cellular iron distribution in Bacillus anthracis. J Bacteriol 194(5):932–940. doi:10.1128/jb.06195-11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. van Schaik W, Chateau A, Dillies MA, Coppee JY, Sonenshein AL, Fouet A (2009) The global regulator CodY regulates toxin gene expression in Bacillus anthracis and is required for full virulence. Infect Immun 77(10):4437–4445. doi:10.1128/iai.00716-09

    Article  PubMed Central  PubMed  Google Scholar 

  37. van Schaik W, Prigent J, Fouet A (2007) The stringent response of Bacillus anthracis contributes to sporulation but not to virulence. Microbiology 153(Pt 12):4234–4239. doi:10.1099/mic.0.2007/010355-0

    Article  PubMed  Google Scholar 

  38. Wagner C, Khan AS, Kamphausen T, Schmausser B, Unal C, Lorenz U, Fischer G, Hacker J, Steinert M (2007) Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix. Cell Microbiol 9(2):450–462. doi:10.1111/j.1462-5822.2006.00802.x

    Article  CAS  PubMed  Google Scholar 

  39. Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, Cashel M (1991) Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266(9):5980–5990

    CAS  PubMed  Google Scholar 

  40. Xu Y, Liang X, Chen Y, Koehler TM, Hook M (2004) Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis. J Biol Chem 279(50):51760–51768. doi:10.1074/jbc.M406417200

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded through a grant from the National Research Foundation of Korea (NRF) through the Korean government (MSIP) (No. 2011-0030049).

Author information

Authors and Affiliations

  1. Department of Molecular and Life Sciences, Hanyang University, 1271, Ansan, 426-791, Gyeonggi-do, Republic of Korea

    Se Kye Kim, Moon Kyoo Park, Sang Hoon Kim, Kyoung Hwa Jung, Jang W. Yoon & Young Gyu Chai

  2. Department of Bioprocess Technology, BioPolytechnic College, Nonsan, 320-905, Republic of Korea

    Kwang Gun Oh

  3. College of Veterinary Medicine, Kangwon National University, Chuncheon, 200-701, Republic of Korea

    Chong-Hae Hong & Jang W. Yoon

  4. Department of Nanobiotechnology, Hanyang University, Seoul, 133-791, Republic of Korea

    Young Gyu Chai

Authors
  1. Se Kye Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Moon Kyoo Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sang Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kwang Gun Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kyoung Hwa Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chong-Hae Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jang W. Yoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Young Gyu Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jang W. Yoon or Young Gyu Chai.

Additional information

Se Kye Kim and Moon Kyoo Park equally contributed to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, S.K., Park, M.K., Kim, S.H. et al. Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog, Rsh Bant . Curr Microbiol 69, 436–444 (2014). https://doi.org/10.1007/s00284-014-0606-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-014-0606-8

Keywords

Search

Navigation