Skip to main content
SpringerLink
Log in

Regulatory Characteristics of Bacillus pumilus Protease Promoters

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5′ regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3–19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis.

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. Balaban NP, Mardanova AM, Sharipova MR, Gabdrakhmanova LA, Sokolova EA, Rudenskaya GN, Leshchinskaya IB (2004) Purification and characterization of serine proteinase 2 from Bacillus intermedius 3–19. BioChemistry 69:420–426. doi:10.1023/B:BIRY.0000026198.81752.f4

    CAS  PubMed  Google Scholar 

  2. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bisicchia P, Botella E, Devine KM (2010) Suite of novel vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the amyE and bglS loci in Bacillus subtilis. Plasmid 64:143–149. doi:10.1016/j.plasmid.2010.06.002

    Article  CAS  PubMed  Google Scholar 

  4. Botella E, Fogg M, Jules M, Piersma S, Doherty G, Hansen A, Denham EL, Le Chat L, Veiga P, Bailey K, Lewis PJ, van Dijl JM, Aymerich S, Wilkinson AJ, Devine KM (2010) pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis. Microbiology 156:1600–1608. doi:10.1099/mic.0.035758-0

    Article  CAS  PubMed  Google Scholar 

  5. Chai Y, Kolter R, Losick R (2010) Reversal of an epigenetic switch governing cell chaining in Bacillus subtilis by protein instability. Mol Microbiol 78:218–229. doi:10.1111/j.1365-2958.2010.07335.x

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Chai Y, Norman T, Kolter R, Losick R (2010) An epigenetic switch governing daughter cell separation in Bacillus subtilis. Genes Dev 24:754–765. doi:10.1101/gad.1915010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chumsakul O, Takahashi H, Oshima T, Hishimoto T, Kanaya S, Ogasawara N, Ishikawa S (2011) Genome-wide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abh reveals their interactive role in transcriptional regulation. Nucleic Acids Res 39:414–428. doi:10.1093/nar/gkq780

    Article  CAS  PubMed  Google Scholar 

  8. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33–38

    Article  CAS  PubMed  Google Scholar 

  9. Danilova YV, Shagimardanova EI, Margulis AB, Toymentseva AA, Balaban NP, Rudakova NL, Rizvanov AA, Sharipova MR, Palotás A (2014) Bacterial enzymes effectively digest Alzheimer’s β-amyloid peptide. Brain Res Bull 108:113–117. doi:10.1016/j.brainresbull.2014.08.009

    Article  CAS  PubMed  Google Scholar 

  10. Ferrari E, Jarnagin AS, Schmidt BF (1993) Commercial production of extracellular enzymes. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other Gram-positive bacteria. American Society for Microbiology, Washington, DC, pp 917–937

    Google Scholar 

  11. Fujita M, Losick R (2005) Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A. Genes Dev 19:2236–2244. doi:10.1101/gad.1335705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gabdrakhmanova L, Vishniakov I, Sharipova M, Balaban N, Kostrov S, Leshchinskaya I (2005) Salt stress induction of glutamyl endopeptidase biosynthesis in Bacillus intermedius. Microbiol Res 160:233–242. doi:10.1016/j.micres.2004.05.005

    Article  CAS  PubMed  Google Scholar 

  13. Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32. doi:10.1007/s00253-002-0975-y

    Article  CAS  PubMed  Google Scholar 

  14. Harwood CR, Cutting SM (1990) Molecular Biological Methods for Bacillus. Wiley, Chichester

    Google Scholar 

  15. Itskovich EL, Liutova LI, Balaban NP, Mardanova AM, Shakirov EV, Sharipova MR, Leshchinskaia IB, Rudenskaia GN (1998) Thrombolytic and anticoagulant properties of thiol-dependent proteinase from Bacillus intermedius 3–19. Vopr Med Khim 44:288–291

    CAS  PubMed  Google Scholar 

  16. Jordan S, Rietkötter E, Strauch MA, Kalamorz F, Butcher BG, Helmann JD, Mascher T (2007) LiaRS-dependent gene expression is embedded in transition state regulation in Bacillus subtilis. Microbiology 153:2530–2540. doi:10.1099/mic.0.2007/006817-0

    Article  CAS  PubMed  Google Scholar 

  17. Kaiumov AR, Sabirova AR, Balaban NP, Mardanova AM, Il’inskaia ON, Kostrov SV, Sharipova MR (2008) Start codon in the serine proteinase gene from Bacillus intermedius. Mol Biol (Mosk) 42:117–122. doi:10.1007/s11008-008-1015-5.

    Article  CAS  Google Scholar 

  18. Kallio PT, Fagelson JE, Hoch JA, Strauch MA (1991) The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein. J Biol Chem 266:13411–13417

    CAS  PubMed  Google Scholar 

  19. Kayumov AR, Kirillova JM, Mikhailova EO, Balaban NP, Sharipova MR (2006) The prediction of regulation of subtilisin-like proteinase gene from Bacillus intermedius through its regulatory sequence analysis. BGRS-2006: Proceedings of the fifth international conference on Bioinformatics of Genome Regulation and Structure, Novosibirsk, July 16–22, pp 65–68.

  20. Kearns DB, Losick R (2005) Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev 19:3083–3094. doi:10.1101/gad.1373905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kobayashi K (2007) Gradual activation of the response regulator DegU controls serial expression of genes for flagellum formation and biofilm formation in Bacillus subtilis. Mol Microbiol 66:395–409. doi:10.1111/j.1365-2958.2007.05923.x

    Article  CAS  PubMed  Google Scholar 

  22. Kodgire P, Dixit M, Rao KK (2006) ScoC and SinR negatively regulate epr by corepression in Bacillus subtilis. J Bacteriol 188:6425–6428. doi:10.1128/JB.00427-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kunst F, Rapoport G (1995) Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177:2403–2407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mäder U, Antelmann H, Buder T, Dahl MK, Hecker M, Homuth G (2002) Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics. Mol Genet Genom 268:455–467. doi:10.1007/s00438-002-0774-2

    Article  Google Scholar 

  25. Mikhailova EO, Mardanova AM, Balaban NP, Rudenskaya GN, Ilyinskaya ON, Sharipova MR (2009) Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth. BioChemistry 74(3):308–315. doi:10.1134/S0006297909030109

    CAS  PubMed  Google Scholar 

  26. Mirouze N, Prepiak P, Dubnau D (2011) Fluctuations in spo0A transcription control rare developmental transitions in Bacillus subtilis. PLoS Genet 7(4):e1002048. doi:10.1371/journal.pgen.1002048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Msadek T (1999) When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis. Trends Microbiol 7:201–207

    Article  CAS  PubMed  Google Scholar 

  28. Msadek T, Kunst F, Klier A, Rapoport G (1991) DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J Bacteriol 173:2366–2377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ogura M, Matsuzawa A, Yoshikawa H, Tanaka T (2004) Bacillus subtilis SalA (YbaL) negatively regulates expression of scoC, which encodes the repressor for the alkaline exoprotease gene, aprE. J Bacteriol 186:3056–3064. doi:10.1128/JB.186.10.3056-3064.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ogura M, Shimane K, Asai K, Ogasawara N, Tanaka T (2003) Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis. Mol Microbiol 49:1685–1697. doi:10.1046/j.1365-2958.2003.03665.x

    Article  CAS  PubMed  Google Scholar 

  31. Ohsawa T, Tsukahara K, Ogura M (2009) Bacillus subtilis response regulator DegU is a direct activator of pgsB transcription involved in gamma-poly-glutamic acid synthesis. Biosci Biotechnol Biochem 73:2096–2102. doi:10.1271/bbb.9034

    Article  CAS  PubMed  Google Scholar 

  32. Sabirova AR, Rudakova NL, Balaban NP, Ilyinskaya ON, Demidyuk IV, Kostrov SV, Rudenskaya GN, Sharipova MR (2010) A novel secreted metzincin metalloproteinase from Bacillus intermedius. FEBS Lett 584:4419–4425. doi:10.1016/j.febslet.2010.09.049

    Article  CAS  PubMed  Google Scholar 

  33. Sánchez A, Olmos J (2004) Bacillus subtilis transcriptional regulators interaction. Biotechnol Lett 26:403–407

    Article  PubMed  Google Scholar 

  34. Shafikhani SH, Mandic-Mulec I, Strauch MA, Smith I, Leighton T (2002) Postexponential regulation of sin operon expression in Bacillus subtilis. J Bacteriol 184:564–571. doi:10.1128/JB.184.2.564-571.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sharipova M, Balaban N, Kayumov A, Kirillova Y, Mardanova A, Gabdrakhmanova L, Leshchinskaya I, Rudenskaya G, Akimkina T, Safina D, Demidyuk I, Kostrov S (2008) The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis. Microbiol Res 163:39–50. doi:10.1016/j.micres.2006.03.003

    Article  CAS  PubMed  Google Scholar 

  36. Sharipova MR, Shagimardanova EI, Chastukhina IB, Shamsutdinov TR, Balaban NP, Mardanova AM, Rudenskaya GN, Demidyuk IV, Kostrov SV (2007) The expression of Bacillus intermedius glutamyl endopeptidase gene in Bacillus subtilis recombinant strains. Mol Biol Rep 34:79–87. doi:10.1007/s11033-006-9017-7

    Article  CAS  PubMed  Google Scholar 

  37. Sharipova MR, Toymentseva AA, Sabirova AR, Mukhametzianova AD, Akhmetova AI, Mardanova AM, Balaban NP (2011) New phylogenetic position Of Bacillus Intermedius 3–19 strain. Mikrobiologiia 80:424–426. doi:10.1134/S0026261711030192

    CAS  PubMed  Google Scholar 

  38. Stepanov VG, Tirumalai MR, Montazari S, Checinska A, Venkateswaran K, Fox GE (2016) Bacillus pumilus SAFR-032 genome revisited: sequence update and re-annotation. PLoS One 11(6):e0157331. doi:10.1371/journal.pone.0157331

    Article  PubMed  PubMed Central  Google Scholar 

  39. Strauch MA, Hoch JA (1993) Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression. Mol Microbiol 7:337–342

    Article  CAS  PubMed  Google Scholar 

  40. Tsukahara K, Ogura M (2008) Characterization of DegU-dependent expression of bpr in Bacillus subtilis. FEMS Microbiol Lett 280:8–13. doi:10.1111/j.1574-6968.2007.01019.x

    Article  CAS  PubMed  Google Scholar 

  41. Zheng XL (2013) Structure-function and regulation of ADAMTS-13 protease. J Thromb Haemost 1:11–23. doi:10.1111/jth.12221

    Article  Google Scholar 

Download references

Acknowledgements

The authors express their gratitude to Dr. Yevgeniy V. Shakirov, University of Texas at Austin, USA, for reviewing the manuscript. The authors thank Sebastian Hübner for invaluable assistance with setting up the BioTek multimode microtiter plate reader and the corresponding data analysis. We thank Alexey Y. Gorbachev for his generous help with EMSA analysis. We are also grateful to the members of T. Mascher lab for B. subtilis mutant strains used in this study. The reported study was funded by RFBR, according to the research project No. 16-34-60198 mol_а_dk. Some genetic constructions were done within the DAAD-grant “Forschungsstipendien für Doktoranden und Nachwuchswissenschaftler” (325-A/09/84065). Work in the T. Mascher group was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Grant MA2837/1-3). M.R. Sharipova was funded by Russian Science Foundation (RSF Grant No. 16-16-04062). The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

Author information

Authors and Affiliations

  1. Department of Microbiology, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Republic of Tatarstan, 18 Kremlyovskaya St., Kazan, Russian Federation, 420008

    Anna A. Toymentseva & Margarita R. Sharipova

  2. Institute of Microbiology, Technische Universität Dresden, Zellescher Weg 20b, 01217, Dresden, Germany

    Thorsten Mascher

Authors
  1. Anna A. Toymentseva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thorsten Mascher
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Margarita R. Sharipova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anna A. Toymentseva.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 440 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Toymentseva, A.A., Mascher, T. & Sharipova, M.R. Regulatory Characteristics of Bacillus pumilus Protease Promoters. Curr Microbiol 74, 550–559 (2017). https://doi.org/10.1007/s00284-017-1212-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-017-1212-3

Keywords

Search

Navigation