Investigation into the role of chaperones in the secretion of haemolysin from Escherichia coli

Haemolysin is a 107 kDa protein which is secreted independently of the general export pathway. It contains a C-terminal signal which directs its secretion through a trans-envelope translocator, comprised of the proteins HlyB, HlyD and TolC. It was reasonsed that because haemolysin is exported post-translationally it may interact with molecular chaperones to maintain a 'loosely folded' secretion-competent conformation. Investigations carried out indicate that the general export chaperone SecB is not required for the efficient secretion of haemolysin. Preliminary studies using a secB null mutant, in which secretion was significantly reduced, suggested that SecB was essential for efficient secretion. However, further assays using a SecB sequestering approach and complementation of the secB null mutant indicated that SecB is not required either directly, to modulate haemolysin folding, or indirectly in the assembly of the membrane translocator. The reduced secretion by the secB null mutant is probably due to the pleiotropic effects of the mutation. The SecB sequestering approach has also been reproduced in a T7 expression strain using a newly constructed T7 sequesterer. Following further characterisation, this T7 sequestering approach may be used in pulse-chase experiments without the need for immunoprecipitation, enabling the requirements of exported proteins for SecB to be determined in the absence of specific antisera. Further investigations into the possible requirement of chaperones in the secretion of haemolysin have shown the presence of the fully functional chaperonin GroEL to be a strict requirement for efficient secretion of haemolysin. Using a temperature-sensitive groEL mutant, the level of haemolysin secretion was dramatically reduced at both the permissive and non-permissive temperatures

Membrane targeting and insertion of the sensor protein KdpD and the C-tail anchored protein SciP of Escherichia coli

In E. coli, most inner membrane proteins are targeted in a co-translational manner by the universally conserved signal recognition particle (Bernstein et al. 1989; Valent et al. 1998; Schibich et al. 2016). SRP scans the translating ribosomes and binds with high affinity to an exposed SRP signal sequence, present in the nascent chain (Bornemann et al. 2008; Holtkamp et al. 2012; Saraogi et al. 2014). After targeting to the membrane-associated SRP receptor FtsY, the nascent membrane protein is forwarded to the Sec translocase or to the YidC insertase to be integrated into the bilayer (Miller et al. 1994; Cross et al. 2009; Welte et al. 2012; Akopian et al. 2013). In general, the targeting and insertion pathways of inner membrane proteins in E. coli are already well studied. However, there is a special class of proteins, the C-tail anchored proteins with only a few members in E. coli, whose insertion mechanisms are unknown in prokaryotes to date. To study those insertion mechanisms, the C-tail anchored protein SciP was used as a model protein. SciP from the enteroaggregative E. coli is a structural component of the type 6 secretion system and contains a transmembrane domain (TMD) at the extreme C-terminal part from amino acid 184 to 206. This results in a large N-terminal cytoplasmic domain of 183 amino acids. In E. coli, there is another protein, the potassium sensor protein KdpD which shares with SciP the commonality of a large N-terminal cytoplasmic domain. KdpD is a four-spanning membrane protein with the first TMD starting at amino acid position 400. For both proteins, with the TMD being located far away from the cytoplasmic N-terminal part, it was thought that they cannot use the co-translational SRP pathway. However, it was shown that KdpD is targeted co-translationally by SRP and a cytoplasmic targeting signal located between amino acids 22-48 was identified (Maier et al. 2008). In this study it was shown that the C-tail anchored protein SciP is also targeted early during translation by SRP. With fluorescence microscopy studies and sfGFP-SciP fusion constructs, two short hydrophobic regions in the N-terminal cytoplasmic domain (amino acids 12-20 and 62-71) were identified as being important for membrane targeting. With artificially stalled ribosomes exposing each of the targeting signal, microscale thermophoresis meausurements decoded that both signals bind to SRP and to a preincubated SRP-FtsY complex, mimicking the next targeting step. Cysteine-accessibilty assays demonstrated that SciP is the first described protein with two targeting signals since the deletion of one of the hydrophobic regions was compensated by the other remaining one in vivo. To decipher the crucial features of the novel cytoplasmic SRP signal sequences of KdpD and SciP alterations in the signal sequences were analyzed with fluorescence microscopy using sfGFP fusion constructs and microscale thermophoresis measurements using stalled ribosomes. These studies revealed that the novel signal sequences have to exceed a threshold level of hydrophobicity to be recognized and bound by SRP and target sfGFP to the membrane. In addition, three positively charged amino acids in the KdpD SRP signal sequence were identified to promote SRP binding. To characterize the binding mechanism of SRP to the signal sequences, in vitro disulphide cross-linking studies with synthesized KdpD22-48, SciP1-27 and SciP54-85 peptides were performed. All three peptides could be cross-linked to the hydrophobic groove of SRP formed by the M domain, which correlates with the binding of SRP to other substrates. Taken together, the results show that SRP binding is not limited to the TMDs of proteins. SRP is also able to recognize short hydrophobic stretches in the cytoplasmic domain of inner membrane proteins. Cysteine-accessibility assays with the C-tail anchored protein SciP decoded that not only SRP is involved in the delivery pathway but also the insertase YidC. With only 11 amino acids in the periplasmic domain SciP matches with the characteristics of other known YidC only substrates. By extending the C-tail of SciP it was found out that a critical length of 20 amino acids exists and that the exceed of this limit makes the insertion of SciP dependent on the Sec translocase. The studies with the extended C-tails of SciP helped to gain more general information about the YidC dependent insertion of proteins. The results obtained with the protein SciP are first indications about how the insertion of C-tail anchored proteins occurs in E. coli. It is assumed that the SRP system and the insertase YidC compensate the absence of the eukaryotic Get system, responsible for the insertion of eukaryotic tail-anchored proteins.In E. coli werden die meisten integralen Proteine co-translational durch das universell konservierte SRP an die Membran geleitet (Bernstein et al. 1989; Valent et al. 1998; Schibich et al. 2016). SRP scannt translatierende Ribosomen und bindet mit hoher Affinität an eine exponierte SRP Signalsequenz der naszierenden Proteinkette (Bornemann et al. 2008; Holtkamp et al. 2012; Saraogi et al. 2014). Nach dem Transport zum Membran-assoziierten SRP Rezeptor FtsY wird das naszierende Membranprotein an die Sec Translokase oder YidC zur Insertion in den Bilayer übergeben (Miller et al. 1994; Cross et al. 2009; Welte et al. 2012; Akopian et al. 2013). Allgemein sind die Transport- und Insertionswege von Membranproteinen in E. coli bereits sehr gut untersucht. Zu einer speziellen Proteinklasse, mit nur wenigen Proteinen in E. coli, gehören die sogenannten C-tail anchored Proteine, deren Insertionsweg in Prokaryoten bislang jedoch unbekannt ist. Um den Insertionsweg zu erforschen, wurde das C-tail anchored Protein SciP als Modellprotein ausgewählt. SciP aus dem enteroaggregativen E. coli stellt eine strukturelle Komponente des Typ 6 Sekretionssystems dar und besitzt eine Transmembrandomäne (TMD) am C-Terminus von Aminosäure (AS) 184 bis 206. Somit weist SciP eine große N-terminale cytoplasmatische Domäne von 183 AS auf. Ein weiteres E. coli Protein, das Sensorprotein KdpD, teilt mit SciP die Gemeinsamkeit einer großen N-terminalen cytoplasmatischen Domäne. KdpD ist ein vier-spänniges Membranprotein, bei dem die erste TMD nach 400 AS beginnt. Zunächst wurde angenommen, dass die spezielle Topologie dieser beiden Proteine einen co-translationalen SRP-abhängigen Transport ausschließt. Dennoch konnte gezeigt werden, dass KdpD durch ein cytoplasmatisches Transportsignal zwischen den AS 22 und 48 durch SRP erkannt und co-translational an die Membran gebracht wird (Maier et al. 2008). In dieser Studie wurde gezeigt, dass ebenfalls das C-tail anchored Protein SciP bereits zu Beginn der Translation durch SRP an die Membran geleitet wird. Mit Hilfe der Fluoreszenzmikroskopie und sfGFP-SciP Fusionskonstrukten wurden zwei kurze hydrophobe Bereiche in der N-terminalen cytoplasmatischen Domäne (AS 12-20 und 62-71) identifiziert, die wichtig für den Transport sind. Mit translations-pausierten Ribosomen, die jeweils eines der beiden Transportsignale von SciP exponieren, konnte durch Microscale Thermophorese entschlüsselt werden, dass beide Transportsignale während der Translation von SRP und einem SRP-FtsY Komplex gebunden werden. Über Cystein-Modifikationsstudien wurde bestätigt, dass SciP das erste beschriebene Protein mit zwei SRP-Signalen darstellt, da die Deletion eines der beiden Signale durch die Anwesenheit des jeweils anderen in vivo ausgeglichen werden kann. Um die ausschlaggebenden Merkmale der neuartigen cytoplasmatischen SRP-Signalsequenzen von KdpD und SciP zu identifizieren, wurden die Signalsequenzen modifiziert. Die Auswirkungen wurden mit Hilfe von sfGFP-Fusionskonstrukten und der Fluoreszenzmikroskopie sowie mit der Microscale Thermophorese und künstlich pausierten Ribosomen analysiert. Damit wurde gezeigt, dass auch die neuartigen Signalsequenzen ein kritisches Hydrophobizitätslevel überschreiten müssen, um von SRP erkannt und gebunden zu werden und daraufhin zum Transport von sfGFP an die Membran führen. Desweiteren konnte gezeigt werden, dass drei positiv geladene AS in der SRP Signalsequenz von KdpD die SRP Bindung verstärken. Um den Bindemechanismus von SRP an die Signalsequenzen zu charakterisieren, wurden in vitro Disulfid-Crosslinking-Studien mit synthetisierten KdpD22-48, SciP1-27 und SciP54-85 Peptiden durchgeführt. Alle drei Peptide konnten an die hydrophobe Bindetasche der M-Domäne von SRP gebunden werden, was mit der Bindung anderer SRP Substrate übereinstimmt. In dieser Arbeit wurde gezeigt, dass die SRP Bindung nicht auf eine TMD eines Proteins beschränkt ist, sondern dass auch kurze hydrophobe Bereiche in cytoplasmatischen Domänen als SRP Signale agieren können. Cystein-Modifikationsstudien mit dem Protein SciP zeigten, dass nicht nur SRP sondern auch YidC am Insertionsprozess beteiligt ist. Mit nur 11 AS in der periplasmatischen Domäne stimmt diese Charakteristik mit anderen YidC Substraten überein. Durch die Verlängerung der periplasmatischen Domäne von SciP zeigte sich, dass es eine kritische Länge von 20 AS gibt und dass die Überschreitung die Insertion Sec-abhängig werden lässt. Anhand der Studien mit den verlängerten periplasmatischen Domänen von SciP konnten allgemeine Informationen über den YidC Insertionsprozess erlangt werden. Die Ergebnisse mit SciP sind die ersten Anhaltspunkte, wie die Insertion der speziellen C-tail anchored Proteine in E. coli stattfinden könnte. Es wird angenommen, dass das SRP System und die YidC Insertase das Fehlen des eukaryotischen Get-Systems, welches eukaryotische tail-anchored Proteine inseriert, ersetzen kann

Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Directed Evolution Designed to Optimize the in vivo Protein Folding Environment.

Protein folding is assisted by molecular chaperones and folding catalysts in vivo. Understanding how chaperones are regulated and how they function in vivo may provide new avenues for developing protein folding modulators. We used directed evolution which combines DNA manipulation and powerful selection procedures for beneficial mutations in proteins to specifically address these questions. My work focused on two distinct, though closely related problems, both of which have to do with the directed evolution of the periplasmic folding environment of bacteria. My first set of experiments concerned the relationship between the CXXC active site and the functional properties of thiol-disulfide oxidoreductases. Thiol-disulfide oxidoreductases are involved in catalyzing disulfide bond formation, isomerization and reduction during protein folding. We selected for mutants in the CXXC motif of a reducing oxidoreductase, thioredoxin, that complement null mutants in the very oxidizing oxidoreductase, DsbA. We found that altering the CXXC motif affects not only the reduction potential of thioredoxin, but also the ability of the protein to interact with folding protein substrates and reoxidants. Furthermore, the CXXC motif also impacts the ability of thioredoxin to function as a disulfide isomerase. Our results indicate that the CXXC motif has the remarkable ability to confer a large number of very specific properties on thioredoxin related proteins, in addition to their usual roles of regulating redox potentials. The second phase of my work sought to optimize the in vivo folding of proteins by linking folding to antibiotic resistance, thereby forcing bacteria to either effectively fold the selected proteins or perish. Here we were able to show that when Escherichia coli is challenged to fold a very unstable protein, it responds by overproducing a protein called Spy, which increases the steady state level of unstable proteins up to nearly 700-fold. In vitro studies demonstrate that Spy functions as a very effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin flexible cradle-shape dimers with an apolar concave surface, unlike the structure of any previously solved chaperone.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78742/1/shuquan_1.pd

Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Biophysical characterisation of LcrH, a class II chaperone of the type III secretion system

The type three-secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called “translocators”. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats (TPRs). However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultra-centrifugation and equilibrium chemical denaturations showed that LcrH is a weak and thermodynamically unstable dimer (KD ≈ 15 μM, ΔGH2O = 7.4 kcalmol-1). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (ΔGH2O = 1.7 kcalmol-1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O = 5.7 kcalmol-1). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far larger translocator cargo

Structural and functional studies of porins from pathogenic bacteria

Multi-drug resistant bacteria have become a real threat to public health worldwide. Gram-negative bacteria, in particular, have shown high level of antibiotic resistance due to the presence of an additional membrane, known as outer-membrane (OM), that acts as an extra barrier. Most antibiotics enter the cells via a particular class of outer-membrane proteins (OMPs) known as porins. Porins are β-barrel channels that allow the passive diffusion of hydrophobic compounds. The porins are known to select against molecules on the basis of size and charge. When exposed to antibiotics, bacteria can modify the OM permeability by altering their porins profile. Mutations affecting the size and conductivity of the pore channel, and modification of the level of porins expression are just a few examples of how the bacteria can decrease the influx of antibiotics. In order to better understand their interaction with antibiotics, this thesis presents structural and functional studies on porins from pathogenic bacteria. The structure of the natively expressed major outer-membrane protein (MOMP) from Campylobacter jejuni was determines, revelling the presence of a calcium-binding site inside the channel. Electro-physiology and in silico modelling analysis have shown to be important for the stability and the function of the protein. Omp50 from C. jejuni was expressed in E. Coli and its tyrosine kinase activity was analysed in vitro. Finally the structures of the two major porins from Enterobacter aerogens were determined and compared to their orthologs within the Enterobacteriaceae family. Further, a liposome-swelling assay (LSA) was used to deter-mine the rate of permeation of clinically relevant antibiotics through a series of porins. Combining these data allow a more detailed molecular understanding of translocation

Engineering and characterisation of anti-progesterone OBodies

Molecular interactions are fundamental to communication between different parts of the cell or of an organism. These interactions can be weak and transient or strong and semi-permanent. In the case of the adaptive immune system, strong interactions between foreign antigens and specific antibodies lead to a cascade of events comprising the immune response. This phenomenon has been exploited by industry to produce high affinity binding molecules that have been used as therapeutics or diagnostics and engineered antibodies have been at the forefront of these industries. More recently, novel non-immunoglobulin binding proteins, have been similarly engineered to produce high affinity binding proteins that can potentially replace the binding function(s) of antibodies or even surpass them. The OB-fold is a high affinity binding protein domain which has previously been engineered to bind to Hen Egg-white Lysozyme (HEL) with nanomolar affinity as a proof-of-concept technology and has been given the name OBodies. This thesis explores this concept further with an OBody (D7) engineered to bind to the small molecule progesterone (P4) with potential applications to detect P4 in cow’s milk to evaluate pregnancy. Obody-P4 binding was characterised with an optimized ELISA system. This was followed by the engineering of improved versions of this OBody using phage display technology and structural characterisation of one such Obody-P4 complex using X-ray Crystallography. The three-dimensional structure provided surprising insights into the nature of the molecular interactions between P4 and the OBody. During phage display selection a new signal sequence was fortuitously discovered that provided an advantage during phage display. The new signal sequence was investigated and characterised using mutants of GFP and a DARPin sequence to uncover the nature of the selective advantage. The combination of this new signal sequence and thermostable OBody libraries further demonstrates the potential of this system to produce robust bio-sensors for diagnostic and therapeutic applications

A role for proteobacterial mammalian cell entry domains in phospholipid trafficking and infection

Mammalian cell entry (MCE) domains are so called due to the reported ability of an Escherichia coli strain harbouring the mce1 gene from Mycobacterium tuberculosis to invade mammalian cells. Bioinformatic analyses presented here demonstrated that proteins containing a single MCE domain are widespread in bacteria and that proteins containing multiple MCE domains are specific to and have evolved within Proteobacteria. Gene neighbourhood analyses revealed that MCE domain containing proteins are components of transporters and that multi MCE domain containing proteins constitute a novel type of transporter. E. coli was shown to harbour three MCE proteins: the single MCE domain protein MlaD and two multi-domain proteins PqiB and YebT. All three proteins were shown to locate to the inner membrane and bind phospholipids. Phenotypic studies revealed that their functions overlap but are distinct. Infection studies with Salmonella showed that the proteins are important for systemic infection but are not required for mammalian cell entry. Phospholipid growth experiments with Salmonella demonstrated that they are important for phospholipid uptake. These findings suggest that MCE domain containing proteins in Proteobacteria are not directly involved in mammalian cell entry and instead play a role in other aspects of mammalian infection related to phospholipid trafficking

Substrate targeting and recognition in the flagellar type III secretion pathway

The bacterial flagellum is a complex molecular machine that enables bacteria to move as well as having additional roles in biofilm formation, adhesion and host cell invasion. The sequential assembly of the flagellar rod, hook and filament requires the export of thousands of structural subunits across the bacterial cell membrane by a specialised flagellar type III secretion system (fT3SS) located at the base of each flagellum. These subunits are unfolded and exported into a narrow 20Å wide channel in the centre of each flagellum energised by the proton motive force (PMF) and facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. Unfolded subunits transit the length of the channel to the flagellum tip where they fold into the nascent structure. The order of subunit export is determined by the stage of flagellum assembly. Early subunits are exported to assemble the rod and hook substructures after which a substrate specificity switch in the fT3SS allows late subunit export. The first aim of this study was to determine how the export signals that reside within early flagellar subunits contribute to export. In this thesis it was found that early flagellar subunits dock at the cytoplasmic domain of FlhB to correctly position an export signal at the subunit extreme N-terminus. The distance between the extreme N-terminal signal and the FlhB gate recognition motif (GRM) was found to be critical for subunit export. Evidence is presented to show that the extreme N-terminal signal of early flagellar subunits converts the FliPQR components of the export gate from a closed to open conformation, allowing subunits to enter the central channel within the flagellum. Further evidence was presented to show that the FlhA component of the flagellar export machinery is involved in export gate opening. Increasing the proton-motive force (PMF) improved subunit export by a strain encoding a FlhA variant defective in export gate opening, indicating that the PMF energises opening of the export gate. The data are compatible with the view that early subunits dock at the cytoplasmic domain of FlhB to correctly position the extreme N-terminal export signal to trigger PMF-driven opening of the export gate. The late filament structural subunits are delivered from their site of synthesis in the cytoplasm to the flagellar export machinery at the membrane by their cognate chaperones. Using motility and export assays, and in vitro and in vivo affinity chromatography pull down assays, data are provided that build on what is currently known about the sequence of binding events between chaperoned subunits and the flagellar export machinery. Specifically, evidence is presented to show that the FlgN chaperone binds the FliJ component of the ATPase complex after FlgN has docked and been released from the cytoplasmic domain of FlhA. The final aim of this study was to determine whether the flagellar export chaperones regulate activation of the PMF-driven fT3SS export machinery. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. This thesis showed that the flagellar export chaperones regulate FliJ activation of FlhA. The data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.Department of Patholog