Characterisation of the twin-arginine protein translocation pathway of Bacillus subtilis [Elektronische Ressource] / von: Ioan Ovidiu Pop

Ovipop

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2004
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Characterisation of the twin-arginine protein
.translocation pathway of Bacillus subtilis
Dissertation
zur Erlangung des akademisches Grades doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena
von: Diplombiologe Ioan Ovidiu Pop
geboren am 08.12.1973 in NasaudGutachter: 1)……………………….
2)……………………….
3)……………………….
2Table of contents
1. Introduction...............................................................................................................................5
1.1 The genus Bacillus.................................................................................................................5
1.2 Protein transport across the bacterial cytosolic membrane.........................6
1.2.1 Cellular compartmentation in bacteria.............6
1.2.2 The ABC transport system…………………………………………………………………..8
1.2.3 The general secretory pathway………………………………………………………………9
1.2.3.1 The general secretory pathway of Escherichia coli................................…………….9
1.2.3.2 The general secretory pathway in Bacillus subtilis...................11
1.2.4 The Tat protein translocation pathway……………………………………………………..13
1.2.4.1 DpH translocation system in plant thylakoids...........................................................13
1.2.4.2 The Tat system of E. coli .......................................................15
1.2.4.2.1 Tat targeting motifs..........................15
1.2.4.2.2 The substrates for twin-arginine translocation in E. coli .....................................17
1.2.4.2.3 The components of E. coli twin-arginine translocation system ............................17
1.2.4.2.4 The mechanism of twin-arginine translocation in E. coli....20
1.2.4.3 The Tat system in Archaea ....................................................................................22
1.2.4.4 The Tat system of B. subtilis..................23
1.2.4.4.1 The signal peptides and the substrates of Tat-dependent export system of B. subtilis
....................................................................................................................................23
1.2.4.4.2 The components of the Tat pathway..23
1.2.4.4.3 The mechanism of the Tat pathway...24
1.3 The scope of the thesis.........................................................................................................25
1.4 Publications included in the cumulative dissertation...............................27
Published articles:………………………………………………………………………………..29
1. The twin-arginine signal peptide of PhoD and the TatA /C proteins of Bacillus subtilis formd d
an autonomous Tat translocation system. ...........................................................................29
2. Sequence specific binding of prePhoD to soluble TatA complexes indicates proteind
mediated targeting of the Tat export in Bacillus subtilis......................................................35
3. Tat-dependent export of E. coli phytase AppA by using the PhoD-specific transport system
of Bacillus subtilis............................................................................................................44
Submitted articles: ……………………………………………………………………………….53
4. Dual localisation of homo-multimeric TatA elucidates its function in Tat-dependent protein
targeting...........................................................................................................................53
32. Results and discussion .............................................................................................................74
2.1 The phosphodiesterase PhoD is a substrate of the B. subtilis Tat export system........................74
2.1.1 The wild type PhoD is not transported by the E. coli Tat system………..…..…………….74
2.1.2 Replacement of Tat signal peptide by a Sec signal peptide results in Sec-dependent export
of PhoD…………………………………………………………….…………………………….75
2.1.3 The signal peptide of PhoD canalises the export to the Tat system………………….….…76
2.1.4 The signal peptide of PhoD canalises the export of E. coli phytase in B. subtilis…….…...77
2.2 Components of B. subtilis Tat pathway.................................................................................78
2.2.1 TatA is a protein with dual topology…………………………….………………………..78d
2.2.2 TatA operates as targeting factor for PhoD…………….…………………………………80d
2.2.3 TatA has a sequence-specific affinity with the signal peptide of PhoD…………………..81d
2.2.4 Cytosolic TatA is organised in soluble homo-oligomers..………………………………...82d
2.2.5 PrePhoD has affinity to TatA homo-oligomeric complexes…..…………………………..82d
2.2.6 Soluble TatA integrates in hydrophobic membranes………..…………………………….83d
2.2.7 TatC is a genuine membrane protein with potential receptor function……………………84d
2.3 The role of type I signal peptidases of B. subtilis in the processing of PhoD.............................86
2.4 The mechanism of the Tat export of B. subtilis ......................................................................87
Summary.....................................................................90
Zusammenfassung .......................................................................................................................91
References...................................................................................................................................93
Selbständigkeitserklärung......... 102
Curriculum Vitae ...................................................................................................................... 103
Acknowledgments:.................... 105
41 Introduction
1.1 The genus Bacillus
The genus Bacillus is one of the most ubiquitous and diverse group of bacteria, with species
being spread in the soil and associated water sources such as rivers, coastal waters (seaside), and
estuaries (Harwood, 1992). The representatives of the genus are rod-shaped, Gram positive bacteria,
characterised by their ability to produce robust endospores in response to adverse environmental
conditions. Although most bacilli are harmless saprophytes, some species are known pathogens of
humans (B. anthracis and B. cereus) or animals (B. anthracis, B. thuringiensis, B. popilliae). As soil
microorganisms, bacilli species secrete numerous enzymes, enabling them to degrade a variety of
substrates and thus to succeed in a complex and continuously changing environment. The ability of
bacilli to secrete large quantities of proteins directly into the growth medium, coupled with the
possibility to grow in large scale fermentors and their established safety record, make them prime
candidates as “Cell Factories” (Simonen & Palva, 1993; Braun et al., 1999). Some species, including
Bacillus licheniformis, B. amyloliquefaciens and B. subtilis, have already a long commercial history as
GRAS (Generally Regarded as Safe) organisms. Secretory protein production in bacilli is a major
biotechnological tool with a market of over $1 billion per year (see table 1) (van Dijl et al., 2002).
The best studied Bacillus species is Bacillus subtilis, a soil bacterium used as model system for
a large variety of industrial applications. Despite its apparent advantages, the use of B. subtilis for the
production of heterologous proteins has been limited. One important limitation is the synthesis of
several extracellular proteases which cause substantial degradation of secreted foreign proteins (Wu et
al., 1991; Harwood, 1992). Strains lacking all but one of these proteases showed a reduced protease
activity with 99,5% and an improvement in the heterologous product stability (Wu et al., 1991). A
second important restriction in the use of B. subtilis for secretory protein production has been the
limited knowledge about its secretion machinery (Harwood, 1992; van Dijl et al., 2002). These
problems can be resolved by a better understanding of its general physiology, particularly of the
secretion apparatus.
In a medical context, this inoffensive microbe, which produces proteins involved in the
biosynthesis of antibiotics, is a valuable model for the study of pathogenic bacilli, such as B. anthracis
and B. cereus.
5Products from Bacillus species
Enzymes Alkaline phosphatases • Laundry detergents
• Leather industry
Amylases • Starch conversion to high fructose
corn syrup (HFCS)
• Desizing of textiles
• Paper industry
• Detergents
Cellulases • Clearing solutions
Glucanase • Brewing industry
Glucose isomerase • Starch industry
Neutral proteases • Leather industry
• Baking
• Aspartame synthesis
Xylanase • Paper industry
Vitamins Riboflavins; Folic acid • Food industry
• Pharmaceutical industry
Insecticides B. thuringiensis • Agriculture
crystal proteins
Table 1. Biotechnological products obtained using Bacillus species.
1.2 Protein transport across the bacterial cytosolic membrane
1.2.1 Cellular compartmentation in bacteria.
Unlike the eukaryotic cells, prokaryots lack subcellular membrane mediated compartmentation.
Still, a few compartments can be distinguished also in bacterial cells.
- The cytosol, is the organic matrix, which is the site for the most important biological
processes in all living cells.
- The cytosolic membrane (or plasma membrane) surrounds the cytosol and is generally made
of a phospholipid bilayer. The membrane usually embeds a large subset of structural proteins a