Abstract
In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abe S, Takayama KI, Kinoshita S (1967) Taxonomical studies on glutamic acid-producing bacteria. J Gen Appl Microbiol 13:279–301
Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746
Berks BC (1996) A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22:393–404
Berks BC, Palmer T, Sargent F (2003) The Tat protein translocation pathway and its role in microbial physiology. Adv Microb Physiol 47:187–254
Billman-Jacobe H, Wang LF, Kortt A, Stewart D, Radford A (1995) Expression and secretion of heterologous proteases by Corynebacterium glutamicum. Appl Environ Microbiol 61:1610–1613
Blaudeck N, Sprenger GA, Freudl R, Wiegert T (2001) Specificity of signal peptide recognition in Tat-dependent bacterial protein translocation. J Bacteriol 183:604–610
Blaudeck N, Kreutzenbeck P, Müller M, Sprenger GA, Freudl R (2005) Isolation and characterization of bifunctional Escherichia coli TatA mutant proteins that allow efficient Tat-dependent protein translocation in the absence of TatB. J Biol Chem 280:3426–3432
Bolhuis A, Tjalsma H, Smith HE, de Jong A, Meima R, Venema G, Bron S, van Dijl JM (1999) Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis. Appl Environ Microbiol 65:2934–2941
Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455
Daffé M (2005) The cell envelope of corynebacteria. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, USA, pp 121–148
Date M, Yokoyama KI, Umezawa Y, Matsui H, Kikuchi Y (2004) High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. J Biotechnol 110:219–226
Date M, Itaya H, Matsui H, Kikuchi Y (2006) Secretion of human epidermal growth factor by Corynebacterium glutamicum. Lett Appl Microbiol 42:66–70
de Keyzer J, van der Does C, Driessen AJM (2003) The bacterial translocase: a dynamic protein channel complex. Cell Mol Life Sci 60:2034–2052
DeLisa MP, Tullman D, Georgiou G (2003) Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway. Proc Natl Acad Sci USA 100:6115–6120
Demchick P, Koch AL (1996) The permeability of the wall fabric of Escherichia coli and Bacillus subtilis. J Bacteriol 178:768–773
Dilsen S, Paul W, Sandgathe A, Tippe D, Freudl R, Thömmes J, Kula RM, Takors R, Wandrey C, Weuster-Botz D (2000) Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system. Appl Microbiol Biotechnol 54:361–369
Eikmanns BJ, Kleinertz E, Liebl W, Sahm H (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 102:93–98
Feilmeier BJ, Iseminger G, Schroeder D, Webber H, Phillips GJ (2000) Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182:4068–4076
Fisher AC, DeLisa MP (2004) A little help from my friends: quality control of presecretory proteins in bacteria. J Bacteriol 186:7467–7473
Freudl R (2005) Staphylococcus carnosus and other Gram-positive bacteria. In: Gellissen G (ed) Production of recombinant proteins. Wiley-VCH, Weinheim, Germany, pp 67–87
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Harwood CR (1992) Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol 10:247–256
Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172
Jensen CL, Stephenson K, Jorgensen ST, Harwood C (2000) Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system. Microbiology (UK) 146:2583–2594
Jongbloed JDH, Martin U, Antelmann H, Hecker M, Tjalsma H, Venema G, Bron S, van Dijl JM, Müller J (2000) TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway. J Biol Chem 275:41350–41357
Jongbloed JDH, Grieger U, Antelmann H, Hecker M, Nijland R, Bron S, van Dijl JM (2004) Two minimal Tat translocases in Bacillus. Mol Microbiol 54:1319–1325
Jongbloed JDH, van der Ploeg R, van Dijl JM (2006) Bifunctional TatA subunits in minimal Tat protein translocases. Trends Microbiol 14:2–4
Kikuchi Y, Date M, Itaya H, Matsui K, Wu LF (2006) Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869. Appl Environ Microbiol 72:7183–7192
Liebl W, Sinskey AJ, Schleifer KH (1992) Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum. J Bacteriol 174:1854–1861
Lindenstrauß U, Brüser T (2006) Conservation and variation between Rhodobacter capsulatus and Escherichia coli Tat systems. J Bacteriol 188:7807–7814
Madsen SM, Beck HC, Ravn P, Vrang A, Hansen AM, Israelsen H (2002) Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme. Appl Environ Microbiol 68:4007–4014
Meens J, Herbort M, Klein M, Freudl R (1997) Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different Gram-positive bacteria. Appl Environ Microbiol 63:2814–2820
Miller JH (1972) A Short Course in Bacterial Genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Peyret JL, Bayan N, Joliff G, Gulik-Krzywicki T, Mathieu L, Schechter E, Leblon G (1993) Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum. Mol Microbiol 9:97–109
Posey JE, Shinnick TM, Quinn FD (2006) Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis. J Bacteriol 188:1332–1340
Quax WJ (1997) Merits of secretion of heterologous proteins from industrial microorganisms. Folia Microbiol 42:99–103
Robinson C, Bolhuis A (2001) Protein targeting by the twin-arginine translocation pathway. Nat Rev Mol Cell Biol 2:350–358
Rygus T, Scheler A, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol 155:535–542
Salim K, Haedens V, Content J, Leblon G, Huygen K (1997) Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum. Appl Environ Microbiol 63:4392–4400
Sambrook J, Fritsch EF Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sandgathe A, Tippe D, Dilsen S, Meens J, Halfar M, Weuster-Botz D, Freudl R, Thömmes J, Kula MR (2003) Production of a human calcitonin precursor with Staphylococcus carnosus: secretory expression and single-step recovery by expanded bed adsorption. Process Biochem 38:1351–1363
Santini CL, Bernadac A, Zhang M, Chanal A, Ize B, Blanco C, Wu LF (2001) Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock. J Biol Chem 276:8159–8164
Schaerlaekens K, Schierova M, Lammertyn E, Geukens N, Anne J, van Mellaert L (2001) Twin-arginine translocation pathway in Streptomyces lividans. J Bacteriol 183:6727–6732
Schäfer A, Schwarzer A, Kalinowski J, Pühler A (1994) Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13022 and analysis of its role in intergeneric conjugation with Escherichia coli. J Bacteriol 176:7309–7319
Schleifer KH, Fischer U (1982) Description of a new species of the genus Staphylococcus: Staphylococcus carnosus. Int J Syst Bacteriol 32:153–156
Simonen M, Palva I (1993) Protein secretion in Bacillus species. Microbiol Rev 57:109–137
Sturm A, Schierhorn A, Lindenstrauss U, Lilie H, Brüser T (2006) YcdB from Escherichia coli reveals a novel class of Tat-dependently translocated hemoproteins. J Biol Chem 281:13972–13978
Thiemann V, Saake B, Vollstedt A, Schäfer T, Puls J, Bertoldo C, Freudl R, Antranikian G (2006) Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii. Appl Microbiol Biotechnol 72:60–71
Thomas JD, Daniel RA, Errington J, Robinson C (2001) Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol 39:47–53
van Dijl JM, Braun PG, Robinson C, Quax WJ, Antelmann H, Hecker M, Müller J, Tjalsma H, Bron S, Jongbloed JDH (2002) Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion. J Biotechnol 98:243–254
van Wely KHM, Swaving J, Freudl R, Driessen AJM (2001) Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev 25:437–454
Acknowledgments
We are very grateful to H. Sahm for his strong support of our work on bacterial protein secretion during now almost 20 years. We thank A. Bida for excellent technical assistance. R. Freudl and J.M. van Dijl were supported in part by European Union Grant LSHG-CT-2004-005257.
Author information
Authors and Affiliations
Corresponding author
Additional information
This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Meissner, D., Vollstedt, A., van Dijl, J.M. et al. Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria. Appl Microbiol Biotechnol 76, 633–642 (2007). https://doi.org/10.1007/s00253-007-0934-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-0934-8