Overexpression of sppA results in the recovery of the BSB1 phenotype in the presence of subtilin.

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Flagella Export Apparatus (FEA)

Bacteria are motile microorganisms. To survive they need to swim or swarm depending on their environment. The bacterial motor that allows this movement is called the flagellum, a rotating semi-rigid helical filament. The flagellum was observed for the first time in 1681, when Antony van Leeuwenhoek, the father of protozoology and bacteriology, observed his own faeces (Dobell 1932). In one of his letters describing this observations, he clearly described a flagellated microscopic parasite named two centuries later as Giardia intestinalis, and wrote that this “little animal” has “sundry little paws” capable of strongly stirring the medium, a clear observation of flagella (Dobell 1932).
The bacterial flagellum is a highly efficient molecular nanomachine with approximately 120-140 nm of diameter and about 10 to 20 μm long, composed by three main structures : the basal body, the hook and the filament, which are made of about 25 different proteins (Erhardt, Namba, and Hughes 2010). The basal body, a membrane-anchored structure that works as a structural anchor, a reversible rotary motor and flagellar protein export system (Minamino, Imada, and Namba 2008). The hook is a curved, thicker and flexible cylinder that joins the basal body to the filament, contributing to the synchronized rotation of the several flagella on the cell (Depamphilis and Adler 1971). The filament is a helical propeller composed by thousands of copies of the protein monomer flagelin (F. Wang et al. 2017). All the components of these protein complexes are exported by the flagellum protein export apparatus, using ATP and PMF as the energy source (Forster and Marquis 2012; Diepold and Armitage 2015). The flagellum is a great example of how membrane protein super-complexes are of importance for cell integrity.
Due to cell envelope differences, Gram-positive and Gram-negative flagellum are structurally different. As the basal body is a transmembrane structure that attaches the filament and, the hook to the cell, it is the structure that has more differences between the two types of bacteria. While the Gram-negative bacteria have a longer basal body composed of four rings – the cytoplasmic disk (C-ring), the membrane disk (M-ring), the periplasm disk (P-ring) and the outer membrane disk (L-ring); the Gram-positive bacteria have only two basal body rings, a M-ring and a P-ring, as shown on Figure 12 (Erhardt, Namba, and Hughes 2010; Mukherjee and Kearns 2014). As I am reviewing the B. subtilis secretion systems, I will describe in detail the flagellar protein export system.

Type IV Secretion Systems

The type IV Secretion System (T4SS) is a long and thin multi-subunit, membrane-spanning translocation system that is present on the surface of many bacterial species. T4SSs have evolved from a self-transmissible, single-stranded DNA conjugation system with VirB4-like AAA + ATPase to systems with an enormous diversity in their overall structure and the types of substrates secreted. Therefore, this pathway serves to translocate a highly diverse family of substrates, since it has a functional astounding collective capacity of (Figure 16 A-E):
 recognizing and translocating single-stranded (ss) DNA substrates across the membrane to bacterial or eukaryotic target cell, whereby direct cell contact is required (conjugation machines).
 delivering effector proteins (effector translocator systems) to the cytosol of eukaryotic target cells in a contact-dependent mechanism.
 exporting/importing molecules from/to the extracellular milieu.
 delivering killing toxins to bacterial neighbours.
 contributing to biofilm development.
Consequently, T4SSs are involved in a variety of functions including pilus formation, toxin and other protein secretion, gene transfer, and biofilm formation (Grohmann et al. 2018). This type of secretion system can be found in Gram-positive as well in Gram-negative bacteria and in some archaea. Furthermore, many pathogenic bacteria use this secretion system as virulence factor and several intracellular symbionts use it for supporting their colonization and propagation in the eukaryotic host (Figure 16 E)(Cascales and Christie 2003).

A bacterial cytoplasmic quality control system: ClpXP protease

Cytoplasmic protease complexes, as ClpXP, are responsible for the degradation of defective or denatured membrane or secretory proteins that, due to stress conditions, cannot be translocated into or out of the membrane and are arrested in the cytoplasm. ClpXP consists of two subunits, the ClpX subunit, which has ATPase activity and form a hexameric ring, and the ClpP, which is a double-heptameric serine protease (Figure 20). The interaction between these proteins was observed for the first time in the early 1990s, Gottesma et al. named and identified E. coli ClpX as the protein that, in the presence of ClpP, induces the ATP-dependent degradation of λ0 protein. They showed that the clpX and clpP genes form an operon and are co-transcribed in a single heat-induced mRNA (Gottesman et al. 1993). As for E. coli ClpP, it has been demonstrated that B. subtilis clpP is also induced by heat shock stress. Additionally, it was shown that B. subtilis clpP is induced by salt and ethanol stress, as well as after a puromycin treatment. Indeed after such a stress exposure the amount of clpP specific mRNA increased; however the highest increase was shown after heat shock stress (Gerth et al. 1998). Therefore, these experiments suggested that B. subtilis ClpP is involved in the cell response to puromycin treatment or heat shock stress, through the degradation of abnormal proteins resulting from the stress. It was also demonstrated that the deletion of clpP or clpX in B. subitilis grown in stress conditions, induced the production of elongated cells (Figure 19) and an impaired growth, indicating that B. subtilis ClpXP have substrates involved in cell morphology or cell division and emphasized the importance of ClpXP as proteases or chaperones in stress tolerance (Gerth et al. 1998).

Membrane-bound proteases, an important quality control step for membrane and secretory proteins.

The bacterial cell envelope, membrane and cell wall, the external layer of bacterial cells, is very important for the integrity and viability of bacteria, as it provides physical protection, determines the cell shape and is the principal stress-bearing element (Scheffers and Pinho 2005; Thomas, Daniel, and Suzanne 2010). These are sophisticated, dynamic and complex structures that, which in addition to protecting, allows selective passage of nutrients from the outside, translocate secretory proteins and waste products from the inside. Gram-negative bacteria cell envelope, such for E. coli, is composed of two membranes, the cytoplasmic membrane and an outer membrane, and between these structures a membrane-enclosed periplasm, a dynamic and metabolically highly active environment. The outer membrane of Gram-negative bacteria contains lipopolysaccharides in its outer leaflet and phospholipids in the inner leaflet. In contrast, Gram-positive bacteria have a cytoplasmic membrane and a cell wall. The cell wall is a complex structure composed of surface proteins, teichoic acids and a thick layer of peptidoglycan (PG). Peptidoglycan, also called murein, is a heteropolymer composed of long glycan chains, made up of alternating β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) subunits, which are cross-linked by peptide bridges to form a strong but flexible structure (ref). Peptidoglycan is present in almost all bacteria, in B. subtilis these glycan strands are considered to be long (between 50 and 250 disaccharide units) when compared to, for instance, Staphylococcus aureus strands (between 3 and 10 disaccharide units), except in Mycoplasma and a few other species that lack detectable cell walls (Vollmer, Blanot, and De Pedro 2008). To recoup the absence of periplasm, Gram-positive bacteria are able to lipo-modify homologues of periplasmic proteins and attach them to the outer leaflet of the cytoplasmic membrane, as well as retain some secretory proteins in the cell wall by ionic or covalent attachment to the peptidoglycan (Sarvas et al. 2004; Schneewind and Missiakas 2014). This way, the Gram-positive cell wall can be described as a thicker but dynamic and flexible structure rich in cell surface proteins, including quality control proteases, extracytoplasmic chaperones, autolysins, surface layer proteins, and substrate binding proteins (Pohl and Harwood 2010a; Schneewind and Missiakas 2014). In addition to peptidoglycan, an important class of cell surface glycopolymers in Gram‐positive bacteria are the phosphate rich teichoic acids (TAs). These molecules play a role in a large variety of functions, such as in maintaining the physicochemical properties of the cell surface, cation homeostasis, resistance to antimicrobial peptides and lytic enzymes, acting as phage receptors, in cell division, biofilm formation and host adhesion. There are two types of TAs, distinguished by the way they are covalently linked to the surface, the lipo-teichoic acids (LTAs), which are anchored to the cytoplasmic membrane, extending from the cell into the peptidoglycan layer, and the wall teichoic acids (WTAs), which are covalently attached to the peptidoglycan layers and extend beyond them. Together, the LTAs and the WTAs, create a negative gradient that goes from the bacterial cell surface until the outer most layers of the PG (C Weidenmaier and Peschel 2008; Pasquina, Santa Maria, and Walker 2013). B. subtilis has two distinct phosphate rich WTAs, the poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate), although the last one is present in minor amounts (Freymond et al. 2006). These anionic polymers and the lipo-teichoic acids create a high density of negative charge in the cell wall, an environment into which secretory proteins emerge in an unfolded state from the translocase (Hyyrylainen et al. 2000).
As described before in this introduction the Gram-positive architecture is compatible to protein export, a closer look at the cell envelope reveals that it offers a very challenging environment for proteins. From the correct insertion of membrane proteins and their maintenance to the unfolded proteins that emerge from the translocase, which should fold into their native configuration quickly without forming intra or intermolecular interactions that could block the translocation machinery and/or block cell wall synthesis sites, either or which would compromise cell viability. This reinforce the importance of having membrane quality control systems. As is the case of HtrA, HtrB and WprA for the Sec pathway, as they are serine proteases, belonging to the quality control protease family, combating the extracellular folding stress, and preventing potentially fatal obstruction of the secretory translocase. Another serine protease of the Sec pathway is the Signal peptide peptidase A (SppA), that will be described in the next subchapter.


Optimisation of the BN-PAGE technique

The BN-PAGE (for Blue Native PAGE) can be used to isolate protein complexes from the membrane fraction, to determine the molecular weight of native protein complex, to obtain information about their oligomeric states and to identify physiological protein–protein interactions (Wittig, Braun, and Schägger 2006). We therefore used the BN-PAGE as a complementary technique to identify the components of membrane protein complexes involved in the Sec pathway. As this electrophoretic technique is performed in native conditions, theoritically, the different protein components of a stable complex are not separated and migrate together in the gel. The n-Dodecyl β-D-maltoside (DDM) was the detergent chosen to solubilize the membrane samples obtained after ultracentrifugation of disrupted B. subtilis cells. After solubilisation (1% DDM) the samples are supplemented with an anionic dye, Coomassie blue G-250, and the protein complexes are separated according to their sizes by electrophoresis on a gradient 4-16% polyacrylamide gel. High molecular weight complexes are running high in the gel. If the concentration of detergent is too high, these complexes dissociate and individual proteins are running lower in the gel.
We used the SecDF-SPA strain to analyse if the BN-PAGE can used for our study as SecDF-SPA is well expressed and can be easily detected by Western-Blot. Again different concentrations of DDM were used to solubilize the SecDF-SPA cell membranes. To obtain better results we have loaded the maximum quantity of protein possible (in this case 17.5μg, all the samples were loaded with same amount) and to avoid contamination between samples, we have loaded the samples with four empty wells between. In BN-PAGE, proteins often appear as a smear and not as single bands as in SDS-PAGE gels (Figure 29). Nonetheless, at low DDM concentrations the smears corresponding to SecDF-SPA appeared higher in the gel, indicating that SecDF-SPA is part of a higher molecular weight complex. In contrast, at higher DDM concentrations this complex is dissociated as SecDF-SPA appeared lower in the gel (Figure 29).

The SppA/YteJ membrane complex

The function of B. subtilis SppA has been studied, as described at the introduction (Signal peptide peptidase A (SppA) section). However, yteJ which in an operon with sppA encodes for a protein of unknown function and unknown structure. As a first step towards the understanding of this unknown protein, we analysed (i) its genomic context using bioinformatics tools, such as BLAST (Singh and Raghava 2016) and microbial genomic context viewer (Overmars et al. 2013), and (ii) its putative structure using structure prediction softwares such as Protter (Figure S 1 of appendix chapter) (Omasits et al. 2014). Using BLAST-P to align the sequence of the Bacillus subtilis sppA yteJ operon with different bacterial genomes of different bacterial species, the results showed that yteJ is often present following sppA. The sppA gene is not only present in B. subtilis but also in most of the bacteria from the firmicutes phylum, including pathogenic bacteria as listeria monocytogenes (Figure 31).

Table of contents :

1.1 General introduction
1.1.1 Project context
1.1.2 Bacillus subtilis, a Gram-positive bacterium
1.1.3 B. subtilis: a Gram-positive model
1.1.4 B. subtilis: a bacterium of industrial interest
1.2 Protein translocation across cytoplasmic membrane in bacteria
1.2.1 The general protein secretion pathway: the Sec system
1.2.2 Substrate recognition
1.2.3 Intracellular chaperoning and targeting to the Sec translocase
1.2.4 Twin-Arginine Transport (Tat) Pathway
1.2.5 Flagella Export Apparatus (FEA)
1.2.6 Type IV Secretion Systems
1.2.7 Holins
1.3 A guarantee of a correct protein translocation in B. subtilis
1.3.1 A bacterial cytoplasmic quality control system: ClpXP protease
1.3.2 Membrane-bound proteases, an important quality control step for membrane and secretory proteins.
1.3.3 Signal peptide peptidase A (SppA)
1.4 Scope of the Thesis
2 Results
2.1 Identification of membrane protein complexes involved in the B. subtilis Sec pathway
2.1.1 SPA-tag constructions
2.1.2 Purifications of the SPA-tagged proteins
2.1.3 Optimisation of the BN-PAGE technique
2.2 The SppA/YteJ membrane complex
2.2.1 The sppA yteJ operon is regulated by sigma factors σA and σW
2.2.2 Deletion of the sppA and yteJ genes
2.2.3 Role of the SppA/YteJ complex in protein secretion
2.2.4 The deletion of sppA yteJ affects the long term survival
2.2.5 The deletion of sppA and yteJ affects the resistance to lantibiotics
2.2.6 The deletions of sppA and yteJ do not affect the resistance to vancomycin, erythromycin or protect against lysozyme
2.2.7 Overexpression of sppA results in the recovery of the BSB1 phenotype in the presence of subtilin.
2.2.8 The overexpression of sppA results in a change of the cell morphology.
2.2.9 SppA and YteJ cell localization
2.2.10 SppA and YteJ in vitro purification.
2.2.11 SppA can digest fully folded proteins or not
2.2.12 YteJ inhibits SppA activity
2.2.13 The C-terminal domain of YteJ is involved in the regulation of SppA activity
2.2.14 SppA digests subtilin
3 Discussion and perspectives
3.1 Protein interactions of the B. subtilis Sec pathway
3.2 SppA/YteJ membrane protein complex
3.2.1 Role of SppA and YteJ in protein secretion:
3.2.2 Role in the resistance to nisin, subtilin and LP9
3.2.3 Regulation of the sppA yteJ operon
3.2.4 SppA/YteJ, a complex involved in quality control of cell division?
3.2.5 The protease activity of SppA and its regulation by YteJ
3.2.6 YteJ, another function?
3.2.7 Model for the interaction of SppA and YteJ
3.3 Main results
4.1 Techniques for DNA manipulation
4.1.1 Oligonucleotide
4.1.2 Polymerase chain reaction (PCR)
4.1.3 Purification of PCR products
4.1.4 Electrophoresis of DNA
4.1.5 Purification of DNA from agarose gel
4.1.6 Purification of plasmid DNA
4.1.7 Chromosomal DNA extraction
4.1.8 Digestion of DNA with restriction enzymes
4.1.9 Ligation of DNA fragments
4.1.10 Gibson assembly technology
4.1.11 Ligation-Independent Cloning (LIC)
4.1.12 DNA sequencing
4.1.13 DNA transformation
4.2 Bacterial strains and growth conditions
4.3 Strain construction
4.3.1 SPA tagged strains construction
4.3.2 Deletion mutants
4.3.3 SppA, YteJ and SppA-YteJ overexpressing strains
4.3.4 Construction of the GFP Fusions
4.3.5 His- tag constructions
4.4 Fluorescence Microscopy
4.5 Live-cell array (LCA)
4.6 Bacteria survival test
4.7 Swimming and swarming capacity
4.8 Biofilm formation
4.9 Protein analysis
4.9.1 Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS PAGE gels)
4.9.2 Blue Native PAGE
4.9.3 Western blot analysis
4.9.4 Secreted proteins analysis
4.9.5 Sequential Peptide Affinity (SPA) purification
4.9.6 His-tag purification
4.9.7 Double tag purification


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