Skip to main content
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2021 Feb 10;26(1):39–46. doi: 10.1007/s12257-020-0397-7

New Bacterial Surface Display System Development and Application Based on Bacillus subtilis YuaB Biofilm Component as an Anchoring Motif

Daeun Kim 1, Wooil Kim 1, Junehyung Kim 1,2,
PMCID: PMC7872719  PMID: 33584103

Abstract

Bacterial surface display system has been adopted in various biotechnological applications. In the case of Bacillus subtilis, most of the studies have been developed using spore based surface display system utilizing the inherent rigidity of spore against heat, alkali, and shear stress. But, spore harvest, purification and separation need additional cost and labor. To eliminate this procedure and to use the gram-positive nature of B. subtilis, YuaB, which is one of the major B. subtilis biofilm components and locates in the cell wall, based cell surface display system, is developed. P43 promoter driven overexpression of YuaB-His6 tag does not hamper bacterial cell growth and promoted biofilm formation of recombinant strain. Flow cytometry of recombinant strain and its protoplast using FITC-Anti His6 antibody, verified that YuaB locate in plasma membrane and protrude to the outside of cell wall, which means YuaB can be used as very efficient anchoring motif. Using surface expressed YuaB-His6 tag, removal of divalent metal ion, Cu2+ and Ni2+, was tried to test its possibility for the environmental application of developed system.

Keywords: YuaB, flow cytometry, Bacillus subtilis, bacterial surface display system, protoplast

Acknowledgement

This work was supported by the Dong-A University research fund.

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Chen X, Gao C, Guo L, Hu G, Luo Q, Liu J, Nielsen J, Chen J, Liu L. DCEO biotechnology: tools to design, construct, evaluate, and optimize the metabolic pathway for biosynthesis of chemicals. Chem. Rev. 2018;118:4–72. doi: 10.1021/acs.chemrev.6b00804. [DOI] [PubMed] [Google Scholar]
  • 2.Clomburg J M, Crumbley A M, Gonzalez R. Industrial biomanufacturing: The future of chemical production. Science. 2017;355:aag0804. doi: 10.1126/science.aag0804. [DOI] [PubMed] [Google Scholar]
  • 3.Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, Boldt J, Khandurina J, Trawick J D, Osterhout R E, Stephen R, Estadilla J, Teisan S, Schreyer H B, Andrae S, Yang T H, Lee S Y, Burk M J, Van Dien S. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat. Chem. Biol. 2011;7:445–452. doi: 10.1038/nchembio.580. [DOI] [PubMed] [Google Scholar]
  • 4.Kim S Y, Yang Y H, Choi K Y. Bioconversion of plant hydrolysate biomass into biofuels using an engineered Bacillus subtilis and Escherichia coli mixed-whole cell biotransformation. Biotechnol. Bioprocess Eng. 2020;25:477–484. doi: 10.1007/s12257-019-0487-6. [DOI] [Google Scholar]
  • 5.Park S, Lee C, Lee J, Jung S, Choi K Y. Applications of natural and synthetic melanins as biosorbents and adhesive coatings. Biotechnol. Bioprocess Eng. 2020;25:646–654. doi: 10.1007/s12257-020-0077-7. [DOI] [Google Scholar]
  • 6.Oh H D, Cho M S, Kim J S, Kim M S, Kim C H, Kang J Y. Identification and characterization of a cocoon degradable enzyme from the isolated strain Bacillus subtilis Bs5C. Biotechnol. Bioprocess Eng. 2020;25:442–449. doi: 10.1007/s12257-019-0399-5. [DOI] [Google Scholar]
  • 7.Jin P, Zhang L, Yuan P, Kang Z, Du G, Chen J. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis. Carbohydr. Polym. 2016;140:424–432. doi: 10.1016/j.carbpol.2015.12.065. [DOI] [PubMed] [Google Scholar]
  • 8.Cho S W, Yang J, Park S, Kim B, Seo S W. Complete genome sequence of lactic acid bacterium Pediococcus acidilactici strain ATCC 8042, an autolytic anti-bacterial peptidoglycan hydrolase producer. Biotechnol. Bioprocess Eng. 2019;24:483–487. doi: 10.1007/s12257-019-0037-2. [DOI] [Google Scholar]
  • 9.Feng J, Gu Y, Quan Y, Cao M, Gao W, Zhang W, Wang S, Yang C, Song C. Improved poly-gamma-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering. Metab. Eng. 2015;32:106–115. doi: 10.1016/j.ymben.2015.09.011. [DOI] [PubMed] [Google Scholar]
  • 10.Lee S Y, Kim H U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 2015;33:1061–1072. doi: 10.1038/nbt.3365. [DOI] [PubMed] [Google Scholar]
  • 11.Gu Y, Xu X, Wu Y, Niu T, Liu Y, Li J, Du G, Liu L. Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications. Metab. Eng. 2018;50:109–121. doi: 10.1016/j.ymben.2018.05.006. [DOI] [PubMed] [Google Scholar]
  • 12.Chen H, Ullah J, Jia J. Progress in Bacillus subtilis spore surface display technology towards environment, vaccine development, and biocatalysis. J. Mol. Microbiol. Biotechnol. 2017;27:159–167. doi: 10.1159/000475177. [DOI] [PubMed] [Google Scholar]
  • 13.Kim J, Schumann W. Display of proteins on Bacillus subtilis endospores. Cell Mol. Life Sci. 2009;66:3127–3136. doi: 10.1007/s00018-009-0067-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Freudl R, MacIntyre S, Degen M, Henning U. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J. Mol. Biol. 1986;188:491–494. doi: 10.1016/0022-2836(86)90171-3. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang Y, Dong W, Lv Z, Liu J, Zhang W, Zhou J, Xin F, Ma J, Jiang M. Surface display of bacterial laccase CotA on Escherichia coli cells and its application in industrial dye decolorization. Mol. Biotechnol. 2018;60:681–689. doi: 10.1007/s12033-018-0103-6. [DOI] [PubMed] [Google Scholar]
  • 16.Plomp M, Carroll A M, Setlow P, Malkin A J. Architecture and assembly of the Bacillus subtilis spore coat. PLoS One. 2014;9:e108560. doi: 10.1371/journal.pone.0108560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Harrold Z R, Hertel M R, Gorman-Lewis D. Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity. J. Microbiol. Methods. 2011;87:325–329. doi: 10.1016/j.mimet.2011.09.014. [DOI] [PubMed] [Google Scholar]
  • 18.Waller L N, Fox N, Fox K F, Fox A, Price R L. Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis. J. Microbiol. Methods. 2004;58:23–30. doi: 10.1016/j.mimet.2004.02.012. [DOI] [PubMed] [Google Scholar]
  • 19.Liew P X, Wang C L C, Wong S L. Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display. J. Bacteriol. 2012;194:161–175. doi: 10.1128/JB.05711-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Nguyen H D, Phan T T P, Schumann W. Analysis and application of Bacillus subtilis sortases to anchor recombinant proteins on the cell wall. AMB Express. 2011;1:22. doi: 10.1186/2191-0855-1-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.López D, Kolter R. Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis. FEMS Microbiol. Rev. 2010;34:134–249. doi: 10.1111/j.1574-6976.2009.00199.x. [DOI] [PubMed] [Google Scholar]
  • 22.Flemming H C, Wingender J. The biofilm matrix. Nat. Rev. Microbiol. 2010;8:623–633. doi: 10.1038/nrmicro2415. [DOI] [PubMed] [Google Scholar]
  • 23.Whitchurch C B, Tolker-Nielsen T, Ragas P C, Mattick J S. Extracellular DNA required for bacterial biofilm formation. Science. 2002;295:1487. doi: 10.1126/science.295.5559.1487. [DOI] [PubMed] [Google Scholar]
  • 24.Kolodkin-Gal I, Elsholz A K W, Muth C, Girguis P R, Kolter R, Losick R. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase. Genes Dev. 2013;27:887–899. doi: 10.1101/gad.215244.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Cairns L S, Marlow V L, Bissett E, Ostrowski A, Stanley-Wall N R. A mechanical signal transmitted by the flagellum controls signalling in Bacillus subtilis. Mol. Microbiol. 2013;90:6–21. doi: 10.1111/mmi.12342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Chung J D, Stephanopoulos G, Ireton K, Grossman A D. Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation. J. Bacteriol. 1994;176:1977–1984. doi: 10.1128/jb.176.7.1977-1984.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Dietrich L E P, Teal T K, Price-Whelan A, Newman D K. Redox-active antibiotics control gene expression and community behavior in divergent bacteria. Science. 2008;321:1203–1206. doi: 10.1126/science.1160619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wilking J N, Zaburdaev V, De Volder M, Losick R, Brenner M P, Weitz D A. Liquid transport facilitated by channels in Bacillus subtilis biofilms. Proc. Natl. Acad. Sci. USA. 2013;110:848–852. doi: 10.1073/pnas.1216376110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Branda S S, González-Pastor J E, Ben-Yehuda S, Losick R, Kolter R. Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 2001;98:11621–11626. doi: 10.1073/pnas.191384198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Branda S S, Chu F, Kearns D B, Losick R, Kolter R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 2006;59:1229–1238. doi: 10.1111/j.1365-2958.2005.05020.x. [DOI] [PubMed] [Google Scholar]
  • 31.Kearns D B, Chu F, Branda S S, Kolter R, Losick R. A master regulator for biofilm formation by Bacillus subtilis. Mol. Microbiol. 2005;55:739–749. doi: 10.1111/j.1365-2958.2004.04440.x. [DOI] [PubMed] [Google Scholar]
  • 32.Lopez D, Vlamakis H, Kolter R. Generation of multiple cell types in Bacillus subtilis. FEMS Microbiol. Rev. 2009;33:152–163. doi: 10.1111/j.1574-6976.2008.00148.x. [DOI] [PubMed] [Google Scholar]
  • 33.Romero D, Vlamakis H, Losick R, Kolter R. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms. Mol. Microbiol. 2011;80:1155–1168. doi: 10.1111/j.1365-2958.2011.07653.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ostrowski A, Mehert A, Prescott A, Kiley T B, Stanley-Wall N R. YuaB functions synergistically with the exopolysaccharide and TasA amyloid fibers to allow biofilm formation by Bacillus subtilis. J. Bacteriol. 2011;193:4821–4831. doi: 10.1128/JB.00223-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Antelmann H, Tjalsma H, Voigt B, Ohlmeier S, Bron S, van Dijl J M, Hecker M. A proteomic view on genome-based signal peptide predictions. Genome Res. 2001;11:1484–1502. doi: 10.1101/gr.182801. [DOI] [PubMed] [Google Scholar]
  • 36.Wang P Z, Doi R H. Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J. Biol. Chem. 1984;259:8619–8625. doi: 10.1016/S0021-9258(17)39775-2. [DOI] [PubMed] [Google Scholar]
  • 37.Kawamura F, Doi R H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 1984;160:442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kim J H, Kim B G. Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production. Biotechnol. Lett. 2001;23:999–1004. doi: 10.1023/A:1010515632509. [DOI] [Google Scholar]
  • 39.Merchante R, Pooley H M, Karamata D. A periplasm in Bacillus subtilis. J. Bacteriol. 1995;177:6176–6183. doi: 10.1128/jb.177.21.6176-6183.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hochuli E, Bannwarth W, Döbeli H, Gentz R, Stüber D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat. Biotechnol. 1988;6:1321–1325. doi: 10.1038/nbt1188-1321. [DOI] [Google Scholar]
  • 41.Asakawa D, De Pauw E. Difference of electron capture and transfer dissociation mass spectrometry on Ni2+-, Cu2+-, and Zn -polyhistidine complexes in the absence of remote protons. J. Am. Soc. Mass Spectrom. 2016;27:1165–1175. doi: 10.1007/s13361-016-1395-z. [DOI] [PubMed] [Google Scholar]
  • 42.Morikawa M. Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species. J. Biosci. Bioeng. 2006;101:1–8. doi: 10.1263/jbb.101.1. [DOI] [PubMed] [Google Scholar]
  • 43.Saffar B, Yakhchali B, Arbabi M. Development of a bacterial surface display of hexahistidine peptide using CS3 pili for bioaccumulation of heavy metals. Curr. Microbiol. 2007;55:273–277. doi: 10.1007/s00284-005-0511-2. [DOI] [PubMed] [Google Scholar]
  • 44.Hinc K, Ghandili S, Karbalaee G, Shali A, Noghabi K A, Ricca E, Ahmadian G. Efficient binding of nickel ions to recombinant Bacillus subtilis spores. Res. Microbiol. 2010;161:757–764. doi: 10.1016/j.resmic.2010.07.008. [DOI] [PubMed] [Google Scholar]
  • 45.Kim W, Kim D, Back S, Lee Y, Abari A H, Kim J. Removal of Ni and Cd by surface display of polyhistidine on Bacillus subtilis spore using CotE anchor protein. Biotechnol. Bioprocess Eng. 2019;24:375–381. doi: 10.1007/s12257-018-0467-2. [DOI] [Google Scholar]
  • 46.Kuroda K, Ueda M, Shibasaki S, Tanaka A. Cell surface-engineered yeast with ability to bind, and self-aggregate in response to, copper ion. Appl. Microbiol. Biotechnol. 2002;59:259–264. doi: 10.1007/s00253-002-1014-8. [DOI] [PubMed] [Google Scholar]
  • 47.Vogt C M, Schraner E M, Aguilar C, Eichwald C. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms. Microb. Cell Fact. 2016;15:137. doi: 10.1186/s12934-016-0532-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Huang G L, Gosschalk J E, Kim Y S, Ogorzalek Loo R R, Clubb R T. Stabilizing displayed proteins on vegetative Bacillus subtilis cells. Appl. Microbiol. Biotechnol. 2018;102:6547–6565. doi: 10.1007/s00253-018-9062-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biotechnology and Bioprocess Engineering are provided here courtesy of Nature Publishing Group

RESOURCES