Verification of structure and function of the ferrous iron-transporter FeoB from Pseudomonas aeruginosa. Master Thesis

Verification of structure and function of the ferrous iron-transporter FeoB from

Pseudomonas aeruginosa

Master Thesis

For the attainment of the academic degree

Master of Science

From the University of Applied Sciences FH Campus Wien

Submitted by:

Andrea Lenger, BSc

Personal identity code 1410544017

Supervisor:

Dr Rietie Venter

University of South Australia Adelaide

Australia

Submitted on:

09.03.2017

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Keywords: FeoB, Iron-transporter, Iron-metabolism, Cystic Fibrosis, Antimicrobial resistance

Pseudomonas aeruginosa is a common bacterium found in many different habitats. It causes diseases in plants and animals, including humans. The pathogen is considered an opportunist as it infects mainly immune- compromised people. The most common diseases are pneumonia in cystic fibrosis patients, tissue infection in burn victims and nosocomial infections.

Infections caused by P. aeruginosa are hard to treat, as the bacteria show a highly intrinsic resistance to antibiotics and other antimicrobial substances.

This natural resistance is mainly attributed to low membrane permeability and caused by very efficient drug-efflux-pumps. These pumps reduce the amount of antimicrobial substances to sub-toxic levels within the cell.

As an opportunist P. aeruginosa does not show high pathogenicity.

Nevertheless, it shows high virulence, which leads to a high infection potential caused by the colonization and rapid growth within the host. This is featured by many virulence-factors. The most noteworthy is the ability of biofilm production. Besides natural resistance, this ability also leads to difficulties in treatment of infections, as the biofilm acts as a physical barrier against antimicrobial substances and cells of the immune system.

For the production of many of these virulence factors, as well as for many different metabolic pathways, iron is an essential substance. This dependence makes the iron-metabolism of pathogens an attractive target in possible treatments of bacterial infections. The uptake of iron is an especially important target, as its blockage would lead to a malfunction in all following processes dependent on iron resulting in starvation and death of the bacteria.

Page 3 of 88 Iron can be present in two different valence states to which bacterial uptake mechanisms have adapted. Hardly soluble ferric iron is taken up in an iron- siderophore-complex. Siderophores are small molecules with a high affinity for ferric iron. In contrast, ferrous iron is highly soluble and is taken up by either the Feo system or Sit system. In P. aeruginosa only the Feo system is known, highlighting its feature as potential target.

The Feo system consists of three proteins, namely FeoA, FeoB and FeoC.

Although the roles of each protein are not completely understood yet, FeoB is known to be the actual permease, which transports iron through the membrane. The natural characteristics of trans-membrane proteins make it hard to study them. Thus, only a few studies on the full length FeoB were conducted.

FeoB is build up by three domains. A N-terminal G-Domain, which hydrolyses GTP and acts as the energy producing compartment of the transporter. The second domain acts as a linker between the G-domain and the C-terminal trans-membrane domain. This domain is supposed to be the permease subunit, acting as the transporter of ferrous iron.

It is believed that this membrane domain builds up a pore of highly conserved cysteines. Another conserved cysteine found on the outside facing side of the protein is supposed to act as an iron sensor, signalling the presence of Fe²⁺ to the other domain and therefore initiating the iron transport.

This project aimes to test the newly developed structure model of the full length FeoB protein. Also the supposed function as an iron transporter will be investigated.

Different methods where used to crosslink the presumed pore-cysteins, indicating the presence of closely located cysteines. Furthermore, it was tested if the cysteines build up a functional pore, which can be opened and closed.

Page 4 of 88 Results of this experiment show a positive outcome, as two different states of the pore could be shown, supposedly an open and a closed state.

To test the function of FeoB as a transporter, the protein was incorporated into the membrane of lipid-vesicles. With the presence of GTP and ferrous iron, the assumed physiological like condition was simulated. Despite a successful incorporation of the protein into the liposomes, no actual transport of iron could be observed.

The aim of this study to support the assumptions of the newly developed structure-model was met, as the results strongly support the accuracy of the model. In contrast, the function of FeoB as an iron transporter could not be shown. Therefore, to fully understand this process additional research has to be done to gain more insight in the mechanism of action. Once the iron transport of the Feo system is fully understood, this knowledge could lead to new developments and strategies for dealing with multi-drug resistant bacteria.

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Schlagwörter: FeoB, Eisen-Transport, Eisen-Stoffwechsel, Mukoviszidose, Antimikrobielle Resistenz

Pseudomonas aeruginosa ist ein weit verbreitetes Bakterium, welches durch seine Anpassungsfähigkeit in verschiedensten Habitaten überleben kann. Es handelt sich hierbei um einen pathogenen Organismus, der verschiedene Krankheitsbilder auslösen kann. P. aeruginosa wird jedoch zu der Klasse der Opportunisten gezählt, da das Bakterium selten Krankheiten in gesunden Individuen auslöst. Durch die starke Präsenz als Umweltkeim ist der Erreger dennoch Hauptursache einiger Infektionen in immun-supprimierten Personen.

Am häufigsten treten Lungenentzündungen bei Patienten mit zystischer Fibrose, Infektionen von Brandwunden, sowie nosokomiale Infektionen auf.

Durch die hohe natürliche Resistenz des Bakteriums gegen verschiedene Antibiotika, sind Pseudomonas Infektionen äußerst schwierig zu behandeln.

Die intrinsische Resistenz ist vor allem bedingt durch eine äußerst niedrige Membran-permeabilität sowie durch effiziente drug-efflux-pumps welche antimikrobielle Substanzen aus dem inneren der Zelle entfernen.

Obwohl P. aeruginosa wie viele Opportunisten eine geringe Pathogenität aufweist, zeigt es eine hohe Virulenz. Diese ermöglicht dem Bakterium eine rasante Besiedelung des Wirtes sowie eine schnelle Vermehrung in ihm.

Verschiedene Faktoren, vor allem jedoch die Fähigkeit Biofilme zu produzieren, ermöglichen diese hohe Virulenz. Diese erschweren die Behandlung, da sie als physische Barriere für antimikrobielle Substanzen und Zellen des Immunsystems fungieren.

Für viele Virulenzfaktoren und verschiedene grundlegen Stoffwechsel-Prozesse ist Eisen essenziell. Diese Abhängigkeit macht den Eisen-Stoffwechsel zu einem attraktiven Ziel im Kampf gegen P. aeruginosa. Vor allem der Prozess

Page 6 of 88 der Eisenaufnahme eignet sich als Angriffspunkt, da alle nachfolgenden Prozesse über ihn reguliert werden. Durch eine Unterbindung der Aufnahme würden die Bakterien schlussendlich aufgrund einer Unterversorgung absterben.

Da Eisen in zwei unterschiedlichen Valenz-Stadien vorliegen kann, haben Bakterien unterschiedliche Verfahren zur Aufnahme entwickelt. Die Aufnahme des beinahe unlöslichen dreiwertigen Eisen Fe(III) erfolgt durch Siderophore, welche eine hohe Affinität für Eisen aufweisen. Die Aufnahme von zweiwertigem Eisen Fe(II) ist bis dato weniger verstanden. Bekannt ist die Aufnahme durch die Systeme Feo und Sit. In P. aeruginosa ist nur das Feo System vorhanden, was die Attraktivität als Angriffspunkt unterstreicht.

Das Feo System wird aus drei unterschiedlichen Proteinen aufgebaut, FeoA, FeoB und FeoC. Die Rolle der einzelnen Proteine ist bisher kaum verstanden, allerdings weiß man, dass FeoB als die konkrete Permease fungiert, welche Fe(II) über die innere Membran transportiert. Durch die natürlichen Eigenschaften von Membranproteinen, die es erschweren sie in vitro zu untersuchen, gibt es nur wenige Untersuchungen zu FeoB. Man weiß jedoch, dass es aus drei Domänen aufgebaut ist.

Die N-terminale cytosolische G-Domäne ist am besten untersucht und ist verantwortlich für die Energieproduktion des Transporters durch GTP- Hydrolyse. Die zweite Domäne fungiert als Verbindungsstück der G-Domäne zur Transmembran-Domäne des Proteins. Dieser Bereich ist vermutlich der eigentliche Transporter für Fe(II).

Es wird angenommen, dass die Transmembran-Domäne eine Pore aus drei hoch konservierten Cysteinen aufbaut. Ein weiteres konserviertes Cystein wurde an der Außenseite der Transmembran-Domäne gefunden. Dieses fungiert vermutlich als Sensor der mit den anderen Domänen kommuniziert, sobald Fe(II) präsent ist.

Page 7 of 88 In diesem Projekt wurde das kürzlich entwickelte Struktur-Modell des vollständigen FeoB Proteins untersucht, sowie dessen Funktionalität als Fe(II)- Transporter.

Mit verschiedenen Methoden wurden die Cysteine der Pore vernetzt um zu überprüfen, ob sie tatsächlich wie angenommen aufgebaut ist. Die Verknüpfung nahe gelegener Cysteine konnte somit gezeigt werden. Weiters wurde die Fähigkeit der Pore sich zu öffnen und zu schließen untersucht. Zu diesem Zweck wurde FeoB mit GTP und Fe(II) inkubiert und mit chemischen Substanzen vernetzt. Die Resultate zeigen, dass die Pore in einem offenen und geschlossenen Zustand vorliegen kann.

Um die Funktionalität als Eisen-Transporter zu untersuchen, wurde FeoB in die Membran eines Lipidvesikels eingebaut und unter der Anwesenheit von GTP und Fe(II) ein annähernd physiologisches Milieu simuliert. Trotz eines erfolgreichen Einbaus konnte kein Transport von Fe(II) nachgewiesen werden.

Ziele dieser Studie waren die Bestätigung des FeoB Struktur-Modells, sowie weitere Untersuchung dessen die Funktionalität. Diese Ziele wurden teilweise erreicht. Die erhaltenen Daten deuten auf die Richtigkeit des Modells hin, die angedachte Funktion des Proteins als Transporter konnte hingegen nicht nach gewiesen werden. Die Ursache dafür, sowie allgemeine weitere Untersuchungen des Proteins sind notwendig, um den Prozess der Fe(II)- Aufnahme vollständig zu verstehen. Dieses Verständnis könnte in weiterer Folge als Basis für verschiedene Behandlungsmöglichkeiten von P. aeruginosa Infektionen dienen.

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Abstract ... 2

Kurzfassung ... 5

Table of Contents ... 8

List of Abbreviations ... 10

1. Introduction ... 12

1.1. Background ... 12

1.2. Resistance ... 13

1.3. Pathogenicity and virulence ... 14

1.4. Biofilm formation ... 15

1.5. Iron requirements in bacteria ... 16

1.6. Uptake of ferrous iron via the Feo System ... 22

1.7. Purpose of this study ... 29

1.8. Aims of the study ... 30

1.9. Significance ... 31

2. Material and Methods ... 32

2.1. Materials ... 32

2.2. Plasmid preparation ... 32

2.3. Competent cells ... 32

2.4. Transformation ... 33

2.5. Inside-Out vesicle preparation... 34

2.6. Protein purification ... 34

2.7. Protein determination ... 35

2.8. SDS-PAGE ... 36

2.9. Western blot... 37

2.10. Crosslinking with dibromobimane ... 38

2.11. Crosslinking of purified FeoB ... 38

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2.12. Crosslinking in ISOV ... 39

2.13. Purification of E. coli phospholipids with Acetone/Ether wash ... 39

2.14. Preparation of proteoliposomes ... 40

2.15. Liposome Assay ... 41

2.16. GTP-/ATP-binding... 42

3. Results ... 43

3.1. Isolation of FeoB expressing plasmids ... 43

3.2. Preparation of Inside Out Vesicles ... 44

3.3. Purification of FeoB ... 46

3.4. GTP-/ATP-binding ... 51

3.5. Crosslinking of pore-lining Cysteines ... 53

3.6. Iron transport ... 65

4. Discussion ... 70

4.1. FeoB expression and purification ... 70

4.2. GTP-/ATP- binding ... 71

4.3. Crosslinking of pore lining cysteines ... 71

4.4. Non fluorescent crosslinker ... 73

4.5. Function as iron transporter ... 76

4.6. Overall outcome ... 78

4.7. Future Outlook ... 78

References ... 80

Acknowledgments ... 85

List of figures ... 86

Independence Declaration ... 88

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ABC ... ATP binding cassette AIDS ... Acquired Immune Deficiency Syndrome APS ... Ammonium persulphate ATP ... Adenosintriphosphat bBBr ... Dibromobimane BMOE ... Bismaleimidoethane BSA ... Bovine serum albumin Cif ... Cystic fibrosis transmembrane conductance regulator inhibitory factor CMC ... Critical micellular concentration Cys ... Cysteine dBBr ... Dibromobimane DDM ... n-Dodecyl-β-D-maltopyranoside DMSO ... Dimethyl Sulphoxide DNA ...Deoxyribonucleic acid DTT ... Dithiothreitol E. coli ... Escherichia coli ECPL ... E. coli phopho lipids EPS ... Extracellular polymeric substance EYPC ... Egg yolk Phosphatidylcholine Fe²⁺ ...Ferrous Iron Fe³⁺ ... Ferric Iron Fur ... Ferric uptake regulator GAP ... GTPase activating protein GDP ... Guanosindiphosphat GTP ... Guanosintriphosphat IPTG ... Isopropyl β-D-1-thiogalactopyranoside ISOV ... Inside out Vesicle kDa ... kilo Dalton K-HEPES ...

4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid pH balanced with potassium hydroxide

KPi ... Phosphate buffer LB ... Lysogeny broth mBBr ... Monobromobimane MEP ... Muciod exoplolysaccharid MOPS ... 3-(N-morpholino)propanesulfonic acid MW ... Molecular weight NFeoB ... N-terminal FeoB – cytoplasmic domain Ni-NTA ... Ni²⁺-nitrilotriacetate resin O/N... Over night OD ... Optical density P. aeruginosa ... Pseudomonas aeruginosa PBP ... Periplasmatic binding protein PCA... phenazine-1-carboxylic acid PEP ... Phosphoenolpyruvate PIPES ... Piperazine-N,N′-bis(2-ethanesulfonic acid) PVDF ... Polyvinylidene fluoride rcf ... Relative centrifugal force

Page 11 of 88 rpm ... Rounds per minute RT ... Roomtemperature SDS ... Sodium dodecyl sulphate SDS-PAGE ... Sodium dodecyl sulphate polyacrylamide gel electrophoresis SOB ... Super optimal broth SOC ... Super optimal broth + 0.5% glucose STD ... Standard TB ... Terrific broth TBST ... Tris buffered saline (TBS with Tween supplementation) TEMED ... Tetramethylethylenediamine TMEA ... Tris(2-maleimidoethyl)amine UTI ... Urinary tract infection WT ... Wild Type

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1 . I n t r o d u c t i o n

1 . 1 . B a c k g r o u n d

Pseudomonas aeruginosa is a Gram-negative, rod-shaped, opportunistic pathogen. These bacteria show a high presence in natural environments, like soil, water, skin flora and most man-made environments throughout the world. Due to this common spread, the pathogen is often causing different diseases in plants and animals, including humans (Baltch, 1994). Persons suffering from P. aeruginosa infections are mainly either immunocompromised or undergoing immunosuppressive treatments (Breidenstein et al., 2011;

Wang et al., 2011), but also immunocompetent persons can get infected. Most common P. aeruginosa infections include urinary tract infections (UTIs), otitis externa ("swimmer’s ear"), hot tub folliculitis, various blood infections and infections of wounds, especially in burn victims (Baltch, 1994). P. aeruginosa is the most common cause for infections in burn victims, as there is a very high degree of exposed skin prior to healing, which is very difficult to protect from pathogens (Lyczak et al., 2000). Also, the pathogen is known for causing secondary infections and being the most common cause for ventilator- associated pneumonia. This complication predominately appears in hospitalised patients, for example AIDS patients and patients who are dependent on live support, like ventilators. Infections with this pathogen are hard to treat, therefore some of the diseases show a high mortality rate of up to 20-30% (Poole, 2011; Rello et al., 1996).

Page 13 of 88 1 . 2 . R e s i s t a n c e

P. aeruginosa shows a high level of intrinsic resistance against many antibiotics (Lambert, 2002). This is due to a variety of different mechanisms, e.g. an effective drug-efflux-pump-system, very low membrane permeability, the ability of target site modification and some other mechanisms.

Additionally, P. aeruginosa is a biofilm producer and can colonize almost any surface. It is a frequent colonizer of medical devices, like catheters, leading to infections or blockage of these devices. It was shown that the pathogen often builds up a biofilm in the lungs of cystic fibrosis patients, causing acute lung inflammation with an often fatal outcome (Høiby et al., 2010). Thus, P. aeruginosa is number one cause of death in cystic fibrosis patients.

The severe impact on this group of patients results in the transition from an aerobic into a mucoid phenotype of the bacteria (Lyczak et al., 2000; Wang et al., 2011). The latter enables the pathogen to survive in the respiratory tract of the patients. Cystic fibrosis is a genetic disorder resulting in a dysfunctional protein which builds up an ion channel. In healthy individuals the channel is responsible for controlled flow of sodium from epithelial cells in the lung and their covering airway surface liquid. A correctly operating channel blocks the sodium inflow to the cells, whereas in patients with cystic fibrosis the channels have lost their function leading to an uncontrolled flow of sodium to the epithelial cells. This ion imbalance causes water flowing into the cells leaving thick mucus on the cell surface, that mucus cannot be effectively removed by the cilia of epithelial cells (Noone and Knowles, 2001; O’Sullivan and Freedman, 2009). Therefore, accumulation of this viscous nutrient-rich mucus occurs, which offers a pleasant habitat for anaerobic pathogens. P. aeruginosa is a facultative aerobic organism which can utilize many different organic materials for nutrition. Thus, it easily inhabits this nutrient rich environment.

The shift in phenotype of the bacteria is also a cause for the production of polysaccharide muciod exopolysaccharid (MEP) which leads to a higher

Page 14 of 88 pathogenicity as it is necessary for bacterial adherence and immune evasion.

80% of cystic fibrosis patients are infected with a chronic P. aeruginosa infection by the age of 3 (Khan et al., 1995), highlighting the severity and common presence of this pathogen.

1 . 3 . P a t h o g e n i c i t y a n d v i r u l e n c e

Pneumonia and other P. aeruginosa related diseases show a very high mortality rate due to the difficult treatment of infections. Thus, these diseases are becoming more and more an enormous issue. In general, opportunistic bacteria are not considered as highly pathogenic, which cannot be held true for P. aeruginosa. Several mechanisms and pathways of the pathogen lead to a high degree of pathogenicity, including inherent resistance to most antibiotics and disinfectants due to low membrane permeability additional to a number of natural and adapted anti-drug mechanisms (Breidenstein et al., 2011; Rello et al., 1996).

The mentioned membrane permeability is 10 to 100 fold less compared to E. coli, highlighting the enormous difficulties with drug treatments (Breidenstein et al., 2011). Since antibiotics and other substances are not transported over the membrane, they have no effect on the bacteria. Another part of intrinsic resistance mechanisms is the continuous expression of efflux pumps, production of antibiotic inactivating enzymes, aminoglycoside modifying enzymes and target site modification (Mesaros et al., 2007). All these factors lead to big issues in the development of novel drugs and treatments of P. aeruginosa infections and highlight the urgent need for the development of new therapies and agents.

Page 15 of 88 1 . 4 . B i o f i l m f o r m a t i o n

Apart from its pathogenicity, P. aeruginosa has a particularly high virulence, causing its high antimicrobial resistance. A major virulence factor is the ability to build biofilms. A biofilm is a matrix of extracellular polymeric substance (EPS) composed of extracellular DNA, proteins and polysaccharides produced by different bacteria (Vu et al., 2009). It can be formed on any given surface, organic or inorganic, and can be found in natural, industrial and hospital environments. The formation of biofilm starts with the attachment of free floating microorganisms to a surface with the help of adhesion factors and pili.

The colony then grows by cell division and recruitment of other bacteria, which are not able to attach to surfaces themselves, but can anchor themselves to the matrix and the already established colony. From this point on the diverse bacterial composition produces polysaccharides to grow a larger biofilm. This stage is called the maturation of a biofilm. If the biofilm and the containing bacterial colony reach a critical size, part of the biofilm disperses and motile bacteria spread to colonize new surfaces. In this late stage, the bacteria produce different enzymes to degrade the biofilm on the outer surface for dispersal (Donlan, 2002).

As soon as bacteria attach to a surface and build up a biofilm, they shift from their planktonic form (i.e. free floating in a fluid environment) to their attached form, which results in production of a different set of virulence factors. The expression of factors supporting a planktonic form, for example motility factors and cytolytic toxins, are decreased, while expression of biofilm virulence factors, like quorum-sensing factors, is increased (Ciofu et al., 2015;

Nadell et al., 2008). The biofilm offers a favourable habitat for many different bacteria. Therefore, many different strains and species can be found in one single biofilm, resulting in a symbiotic relationship between them. The almost impermeable biofilm additionally acts as a barrier against antibiotics, other antimicrobials and the immune system. If a substance is able to penetrate the

Page 16 of 88 biofilm, it is not guaranteed to have an eradicative effect on the bacteria within, as the diffusion rate in the dense matrix is very low (Banin et al., 2005; Wang et al., 2011).

1 . 5 . I r o n r e q u i r e m e n t s i n b a c t e r i a

Iron is an essential element for biofilm formation and many other natural processes. Without iron, thin layers of adherent cells are not able to develop into a fully grown, matured multi-organism biofilm (Banin et al., 2005). Iron has many important roles in P. aeruginosa as it is essential for the production of other expressed and secreted virulence factors contributing to colonization and survival. Examples for iron dependent factors are exotoxin A, endoproteases, pyoverdines (Lamont et al., 2002), alkaline protease and cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif) (Ballok and O’Toole, 2013). Both the latter are involved in pathogenicity of respiratory infection in cystic fibrosis.

Virulence factors have a wide range of effects, including direct toxicity, host- nutrient starving and increased mobility of bacteria, but the main task of these factors is increasing the survival rate of bacteria in the host (Ballok and O’Toole, 2013; Lamont et al., 2002).

Iron is also an essential factor for biofilm formation, as it was shown that iron transport defective mutants were not able to build up a biofilm (Banin et al., 2005). In the case of P. aeruginosa, biofilm formation is strongly linked to infections of lung tissue in cystic fibrosis patients. Besides the already named mutations, these patients often have a deletion of phenylalanine (∆F508)(Davidson and Porteous, 1998). This mutation leads to a higher secretion of iron to the local environment by epithelial cells compared to healthy wild type (WT) cells (Wang et al., 2011).

Page 17 of 88 1.5.1. Role of iron in metabolic pathways

Iron is involved in a large number of cellular processes in all organisms, especially in bacteria. Amongst other things it is important for structural cellular composition, metabolic pathway enzyme activity and a multitude of virulence pathways as described before (Andrews et al., 2003). While environmental bacteria acquire iron from their surroundings, pathogens have to compete with their host in order to obtain iron (Cartron et al., 2006). On top of that, the altered physiological response of the host due to iron- withdrawal causes problems to the pathogens. Due to this variable iron availability and the different valence states in which iron can be present, bacteria developed many different intrinsic iron acquiring and transport mechanisms to guarantee their survival.

Bacteria need a certain amount of iron for their basic physiological functionality. However, excess of iron above this level is used for biofilm formation promoting the transition from motile to sessile form (Andrews et al., 2003; Braun and Hantke, 2011; Messenger and Barclay, 1983; Wang et al., 2011). As iron can have toxic effects when unbound or present in too high concentrations, the intake of iron has to be closely regulated by the organism.

An excess of iron can lead to cell damage by production of hydroxyl free- radicals (Carpenter and Payne, 2014; Kehrer, 2000).

1.5.2. Different valence forms of iron and their uptake pathways

As mentioned above, iron can stably exist in two oxidative forms, ferrous iron (Fe²⁺) and ferric iron (Fe³⁺), which can be found in different environments.

While ferrous iron is soluble, ferric iron is practically insoluble in aquatic solutions (Andrews et al., 2003; Carpenter and Payne, 2014). Due to that,

Page 18 of 88 bacteria developed different cellular influx pathways (Cartron et al., 2006).

While ferric iron is predominately found in aerobic and pH neutral surroundings, ferrous iron is mainly present in acidic and anaerobic environments. Naturally, bacteria focused on a development suitable to the form of iron they are mostly exposed to. Bacteria in the acidic and anaerobic environment of the gastrointestinal tract for example, have to deal with ferrous instead of ferric iron most of the time (Ash et al., 2011). Also biofilms have anaerobic and aerobic sections, so both forms of iron can be present within a biofilm (Lewis, 2001).

As ferrous iron is soluble it is the preferred form for uptake by direct transport of free metal. In contrast, ferric iron has to be chelated or bound to proteins to be transported (Wang et al., 2011). For pathogens another important fact is that ferric iron is always bound to either heam, metalloproteins or ferritin in the host (Crichton and Charloteaux-Wauters, 1987; Skaar, 2010). Therefore, it has to be sequestered before uptake can take place. P. aeruginosa, along with other bacteria, adapted to this variability by developing mechanisms of cellular influx of both forms and iron modification. While being in an aerobic environment the pathogen secretes a reductase (phenazine-1-carboxylic acid, PCA) in order to reduce ferric to ferrous iron and maintaining iron acquisition regardless of the supply or oxygen tension (Cowart, 2002; Wang et al., 2011).

The presence of PCA in the periplasm shows the high preference for reduced iron (Recinos et al., 2012).

1.5.3. Ferric iron uptake mechanisms

As pathogenic bacteria have to compete for iron with host cells or low environmental iron concentrations, they express very efficient iron transport proteins in their membranes as well as the secretion of different proteins to sequester non-free iron (Andrews et al., 2003; Messenger and Barclay, 1983).

Page 19 of 88 To increase the efficiency of these pathways bacteria can utilize them simultaneously. After sequestering ferric iron, proteins solubilise and transport it across the outer membrane (Hantke, 2003; Kim et al., 2012). These transport proteins are also known as siderophores, which are low molecular mass compounds with a high binding affinity for ferric iron (Neilands, 1995;

Wang et al., 2011). P. aeruginosa produces two siderophores, namely pyoverdine and pyochelin (Cornelis and Dingemans, 2013). After binding to the outer cell membrane, the iron-siderophore complex is transported to the cytoplasm via TonB-ExbBD membrane receptors, which consist of a periplasmatic binding protein and an ABC-permease protein complex and transporters. In the cytoplasm, the siderophore is removed from the iron by either reduction of ferric to ferrous iron or siderophore degradation (Carpenter and Payne, 2014; Kim et al., 2012). A scheme of the predominant transport forms can be seen in Figure 1. As siderophores are used by many different species, bacteria developed systems to take over these siderophore-iron complexes specific to other organisms or host cells, P. aeruginosa for example is able to transport fungal siderophores (Carpenter and Payne, 2014).

As iron is such an important survival factor for pathogens, hosts have developed mechanisms to prevent bacteria from iron acquisition, like secreting lactoferrin which has an even higher affinity for iron than siderophores. It is able to sequester iron in the surroundings and prevents growth and biofilm formation (Singh et al., 2002). This effect can also be seen in cystic fibrosis patients. There, iron chelaters like conalbumin are upregulated in the lungs in order to sequester the excess of free iron and inhibit bacterial growth and biofilm formation (Wang et al., 2011).

Figure 1: Essential iron transport systems in membrane receptors (R) bind and transport iron

the cell. Siderophore and haem complexes are transported across the outer membrane, at which point they are bound by periplasmic binding

the cytoplasm through ABC

PBP-siderophore complex through reduction by reductases such as PCA and converted to ferrous iron (Fe²⁺). Ferrous iron is transported from the periplasm through the Feo system however the proposed means o

porin) has yet to be proven. Adapted from

1.5.4. Ferrous uptake mechanisms

Due to heavy inflammation in P. aeruginosa related pneumonia

cell density. This results in a decrease of oxygen tension and a higher presence of iron in the reduced

to ferric iron, it is soluble and can pass the outer membrane without any

Siderophore Siderophore

Essential iron transport systems in Pseudomonas aeruginosa

membrane receptors (R) bind and transport iron-siderophore or iron-haem complexes to e cell. Siderophore and haem complexes are transported across the outer membrane, at which point they are bound by periplasmic binding proteins (PBP) to be transported to the cytoplasm through ABC like transporters. Ferric iron (Fe³⁺) can be liberated from the ex through reduction by reductases such as PCA and converted to ). Ferrous iron is transported from the periplasm through the Feo system however the proposed means of free ferrous iron entry to periplasm (through a porin) has yet to be proven. Adapted from (Carpenter and Payne, 2014)

Ferrous uptake mechanisms

to heavy inflammation in the lungs of cystic fibrosis patients as well as in related pneumonia, the lung tissue shows damage and increased cell density. This results in a decrease of oxygen tension and a higher presence of iron in the reduced ferrous state (Wang et al., 2011)

it is soluble and can pass the outer membrane without any

Siderophore Siderophore-iron-complex

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Pseudomonas aeruginosa. Variable haem complexes to e cell. Siderophore and haem complexes are transported across the outer membrane, ) to be transported to ) can be liberated from the ex through reduction by reductases such as PCA and converted to ). Ferrous iron is transported from the periplasm through the Feo f free ferrous iron entry to periplasm (through a

.

lungs of cystic fibrosis patients as well as in the lung tissue shows damage and increased cell density. This results in a decrease of oxygen tension and a higher (Wang et al., 2011). In contrast it is soluble and can pass the outer membrane without any

Page 21 of 88 additional transport mechanisms (Kim et al., 2012). However, to cross the inner membrane special transport mechanisms are necessary. The two main uptake systems known are called Sit and Feo (Carpenter and Payne, 2014;

Hantke, 2003). While Sit is only present in selective pathogenic bacteria, including all Shigella and some E. coli species, the Feo transport system is predominant in all other bacteria (Boyer et al., 2002; Carpenter and Payne, 2014; Cartron et al., 2006). The schematic mechanism is shown in Figure 1.

1.5.5. Expression control of ferrous iron transport systems

As mentioned above, iron products can be toxic. Therefore, the iron transport is very closely monitored and regulated. The regulation takes place on a genetic level and the control of gene expression is regulated by iron availability. The executive regulator is a transcriptional repressor, namely the ferric uptake regulator (Fur) (Hantke, 2003). It binds as a dimer to Fur boxes as soon as iron uptake starts, resulting in a decreased iron uptake. If iron concentrations are low, a decrease in Fur binding can be observed which leads to expression of genes coding for siderophores, siderophore-ferric iron transporters and ferrous iron transporter (Carpenter and Payne, 2014). Fur acts in a negative feedback loop, meaning it represses its own expression in order to increase uptake of readily available iron. As soon as intercellular levels of iron reach a harmful concentration the redox regulator OxyR induces Fur transcription (Zheng et al., 1999).

Another key player in the regulation of this sensitive system is oxygen. For all facultative aerobic bacteria it is important to sense the presence of oxygen in the surrounding environment, in order to switch either from fermentative to oxidative metabolism or from ferrous to ferric iron transport systems (Carpenter and Payne, 2014; Rolfe et al., 2012). The most common oxygen sensing mechanism utilizes iron in a complex, forming a cluster like [4Fe-4S],

Page 22 of 88 which forms a self-binding dimer and acts as a transcriptional repressor. It is degraded to [3Fe-4S] in the presence of oxygen, leading to loss of function as repressor (Green et al., 2009; Rolfe et al., 2012). However, in low oxygen environments the repressor for siderophore expression is active and additionally acts as an activator for Feo expression (Carpenter and Payne, 2014).

1 . 6 . U p t a k e o f f e r r o u s i r o n v i a t h e F e o S y s t e m

The Feo transport system is encoded on the feoABC operon (structure shown in Figure 2), expressing the three functional proteins FeoA, FeoB and FeoC.

Each protein shows a different structure as well as a different function during the intake process (Hantke, 1987). While FeoA and FeoC are suspected to have independent regulatory functions, FeoB is proposed to be the actual permease subunit (Hantke, 2003; Weaver et al., 2013). Although it was already discovered in 1987 and completely sequenced in 1993, function and structure of all three proteins are yet not fully understood. As the Feo system can be found in the majority of bacterial species it shows a high potential for being an antimicrobial target by starving cells and therefore fight pathogenic infections (Carpenter and Payne, 2014).

Figure 2: Organisation of the

predicted amino acid length of each protein is displayed as well as protein size and the location of the STOP codons of

and Fur boxes for transcriptional regulation, as well as t

potential promoter. Once synthesised the proteins are transported to their cellular locations, FeoA and FeoC to the cytosol and FeoB to the membrane. Adapted from (Cartron et al., 2006).

1.6.1. FeoA

FeoA is a small cytoplasmic protein with an estimated size acids and a molecular weight of 8.3kDa

of FeoA remains unclear not expressed independent

contrast can be expressed independ the presence of an SH3

al., 2006; Kim et al., 2012)

an overall hydrophilic profile, indicating that FeoA is found in the cytoplasm (Cartron et al., 2006). Due to a potential interact

FeoA was assumed to be a GTPase activating protein (GAP

et al., 2012). This link could not be found in previous studies, leav function of FeoA unclear

: Organisation of the feoABC operon in Pseudomonas aeruginosa predicted amino acid length of each protein is displayed as well as protein size and the location of the STOP codons of feoB and feoC. Note the predicted locations of the Fnr and Fur boxes for transcriptional regulation, as well as the upstream location of a potential promoter. Once synthesised the proteins are transported to their cellular locations, FeoA and FeoC to the cytosol and FeoB to the membrane. Adapted from

FeoA is a small cytoplasmic protein with an estimated size of

acids and a molecular weight of 8.3kDa (Kim et al., 2012). The direct function unclear at this point, although it is already known

pressed independently. Instead it is co-expressed with FeoB, e expressed independently (Cartron et al., 2006) the presence of an SH3-domain indicate a FeoA-FeoB interaction al., 2006; Kim et al., 2012). The structure of FeoA shows no N

ic profile, indicating that FeoA is found in the cytoplasm . Due to a potential interaction between FeoA and FeoB, FeoA was assumed to be a GTPase activating protein (GAP) (Dean, 2011; Kim

. This link could not be found in previous studies, leav function of FeoA unclear.

Page 23 of 88

monas aeruginosa. The predicted amino acid length of each protein is displayed as well as protein size and the . Note the predicted locations of the Fnr he upstream location of a potential promoter. Once synthesised the proteins are transported to their cellular locations, FeoA and FeoC to the cytosol and FeoB to the membrane. Adapted from

of about 75 amino . The direct function known that FeoA is expressed with FeoB, which in Cartron et al., 2006). This fact and FeoB interaction (Cartron et . The structure of FeoA shows no N-terminus and ic profile, indicating that FeoA is found in the cytoplasm ion between FeoA and FeoB, (Dean, 2011; Kim . This link could not be found in previous studies, leaving the

Page 24 of 88 1.6.2. FeoB

FeoB is the main functional unit involved in the transport of ferrous iron across the inner membrane. In most bacteria, including P. aeruginosa and E. coli, it is 766 residues long and represents a major virulence factor in many pathogens (Ash et al., 2011; Cartron et al., 2006; Hantke, 2003). Previous studies have shown the essential process of iron transport using radio-labelled 55Fe²⁺ in FeoB deficient bacterial mutants, which were not able to transport ferrous iron (Kammler et al., 1993). However, the direct transport of iron via FeoB still needs to be shown at this point. Also, it is known that some bacteria become attenuated if they lack the Feo system (Hantke, 2003; Stojiljkovic et al., 1993; Tsolis et al., 1996), although some organisms do not show any differences in pathogenicity. This could be due to the Sit transport system also present in these species, which takes over the ferrous iron transport (Cartron et al., 2006).

The structure of FeoB can be distinguished into three regions, each with a specific role in the transport of reduced iron. The N-terminal region (NFeoB) is the best understood and most examined domain of this protein. It is about 160 residues long and a soluble intracellular domain (Marlovits et al., 2002).

The N-terminal region can be subdivided into a GTPase domain and a five- helix domain (helical domain), functioning as a linker to the C-terminal transmembrane domain. The subdomains are shown in Figure 3.

Page 25 of 88

Figure 3: Schematic representation of the domains in FeoB. FeoB consists of a G- domain, a helical domain and a membrane domain. Adapted from (Seyedmohammad et al., 2016).

The structure of the G-domain is super imposable to small Ras-type GTPases in eukaryotes, e.g. p21-Ras and is supposed to be the energy producing region of the protein, as it is capable of binding GTP and hydrolysing it to GDP (Guilfoyle et al., 2014; Hantke, 2003).

Notably, the binding affinity and hydrolysis rates are unusually low compared to the required energy level of comparable iron transporters (Eng et al., 2008;

Kim et al., 2012). This may indicate the presence of either an alternative energy coupling system or a form of local up-regulation system of the GTPase activity.

The GTPase region is built up by five consecutive sequence motifs which are responsible for recognition, coordination and catalysis of GTP. While sequences 1 to 4 are highly conserved, region 5 shows less conservation (Marlovits et al., 2002). Previous studies have shown that region 5 is not involved in GTP

Page 26 of 88 hydrolysis, but maintains GDP release following the hydrolysis (Guilfoyle et al., 2009, 2014). Essential for hydrolysis are motif 2 and 4 (Eng et al., 2008).

Point mutations in these regions, for example D123N mutagenesis, result in incapability of GTP hydrolysis (Marlovits et al., 2002).

Other important structures in this NFeoB domain are Switch I and II consisting of two peptide chains. These structures bind GTP to the G protein region and change their conformation during hydrolysis (Ash et al., 2010). This change is dependent on the bound state of NFeoB. Structural analysis showed that Switch I is important for stabilisation and mediation of GTP hydrolysis, while Switch II does not show an equally important role. However, Switch II is involved in communicating with the helical domain. This signal is assumed to indicate iron transport in the C-terminal domain (Eng et al., 2008;

Wittinghofer and Vetter, 2011).

In contrast to the N-terminal domain, little is known about the C-terminal membrane domain. It assumedly is built up by seven transmembrane alpha- helices, which are highly conserved in all organisms using the Feo transport system (Marlovits et al., 2002). This region is supposed to form the channel for iron transport. The number of helices is unusually high, leading to the assumption that FeoB is both a ferrous iron sensor and transporter (Köster et al.). Within the transmembrane domain, two homologous gates have been identified which are predicted to have opposite orientations. This could be used to transport iron either way, inside and outside of the cytoplasm (Cartron et al., 2006; Köster et al.). Each gate carries a number of highly conserved cysteine residues, which are known to be the best ligands for a soft lewis acid binding, the reaction required for ferrous iron binding (Cartron et al., 2006).

Recently, a structural model of FeoB was constructed (Seyedmohammad et al., 2016). It is predicted to be in a trimeric structure with magnesium being a co-factor for assembly. Each monomer is build up by eight trans-membrane helices. The trimer forms a central pore with the fourth trans-membrane helix.

Page 27 of 88 The pore is lined with three highly conserved cysteine residues (Cys429), one from each monomer. It shows a diameter of 4.5 Å and of 8.5 Å in its opened state. A second highly conserved cysteine residue (Cys675) is located on the seventh trans-membrane helix at the membrane-periplasm interface, but is not involved in any pore building. As it is located on the lipid-facing site it is predicted to act as a ferrous iron sensor, signalling to the G-domain and initiate GTP-binding. A schematic mode of action can be seen in Figure 4.

Figure 4: Schematic representation of the putative mechanism of action of FeoB from P. aeruginosa acting as a GTP-gated Fe²⁺ channel. Fe²⁺ in the periplasm binds to Cys675, which is assumed to act as a Fe²⁺ sensor. The binding signals the G- domain to initiate the binding and hydrolysis of GTP, which results in conformational changes that subsequently open the predicted pore, allowing Fe²⁺ to pass. Adapted from (Seyedmohammad et al., 2016).

This assumption is supported by the results of a GTPase activity testing, which shows a significant lower activity in Cys675 defective mutants. GTP binding and hydrolysis initiate structural changes resulting in an opening of the pore, enabling a flow of ferrous iron. This sensor mechanism has not been identified in any other transporter.

The full homology model of FeoB is shown in Figure 5.

Page 28 of 88

Figure 5: Homology model of FeoB from P. aeruginosa. The subunits are shown in green, red and blue. The GTP ligands are in stick representation. The FeoB homotrimer is viewed either from the plane of the membrane (A) or from the extracellular side of the membrane (B). The central pore is built up by one cysteine (C429) from each of the three monomers, as can be seen in the close up from the extracellular side (C). The orange dashed lines indicate the distance of 8.5 Å between the cysteine residues.

Adapted from (Seyedmohammad et al., 2016).

Page 29 of 88 1.6.3. FeoC

FeoC is a small protein with a length of 80 residues and is the least known and understood part of the Feo transport system. This is due to the little presence in the feoABC operon, which is about 15% only (Hsueh et al., 2013). The structure of FeoC is described as winged-helix protein, which contains four highly conserved cysteine residues. It possibly maintains a [4Fe-4S] cluster, which is necessary for oxygen and iron sensing to avoid iron toxicity as described before. The structure is typical for DNA binding proteins and indicates a function as transcriptional regulator (Hsueh et al., 2013; Kim et al., 2013).

1 . 7 . P u r p o s e o f t h i s s t u d y

After extensive literature research, it is obvious that many questions about the FeoABC system still remain unanswered. The vast majority of studies and research groups focus solely on NFeoB. This may be problematic once the results are transferred to full-length protein function, as the structure of full length proteins can show a different function in comparison to the function of the prevailing sub-domains. This was the case for the comparison of roles of Switch I in NFeoB to full length protein (Marlovits et al., 2002). Still previous research findings can be taken as foundation when developing studies of the full length FeoB. Though the biggest issue in investigating full length FeoB is the insolubility of the membrane domain, which is a common problem with membrane proteins that is hard to address. The natural environment has to be taken into consideration when choosing the solubilising detergent regarding the question whether or not it should contain lipids. It has to be kept in mind that detergents always influence the experiment, even if the chemical behaviour is excellent and the critical micellular concentration (CMC) is reached. Especially when performing protein determination assays could this

Page 30 of 88 become an enormous issue (Seddon et al., 2004). Another complication often occurring with membrane proteins is the low yield of purified protein (Rajarathnam and Rösgen, 2014). This is due to hydrophobic regions, resulting in difficulties with manipulating membrane proteins.

Although studies on FeoA and FeoC were carried out, their role in iron transport is yet unknown. In case they have regulatory functions, their target sites need to be discovered, as well as the underlying mode of action. Whilst the key role of FeoB is known to be involved in iron transport (Kammler et al., 1993), the actual transport of iron by FeoB has not been shown yet. At this point it is not known if there is a direct transport or if Feo acts as a signalling molecule or mediator only. The new homology model (Seyedmohammad et al., 2016) supplies reasonable solutions for the structure which transports ferrous iron across the inner membrane. The model of trimers building a cysteine lined pore has yet to be confirmed.

1 . 8 . A i m s o f t h e s t u d y

As there are various unknown aspects of the Feo transport system, it is hard to decide where to start. The recently developed model of FeoB (Seyedmohammad et al., 2016) leads to a variety of possible further research, provided it can be verified. Thus, the first aim of this study will be to confirm the predicted structure of FeoB. It is assumed that the most important part of FeoB is its predicted central pore, lined by conserved cysteine residues.

Several facts about this pore will be investigated: i) is the pore is actually lined with three cysteins, as suggested by previous studies, ii) are we looking at an actual pore or is it only a structural coincidence of three closely located and iii) do the assumed co-factors have to be combined to initiate the opening of the pore or if one co-factor alone is sufficient to show this effect.

Page 31 of 88 The second aim will be the investigation of key role of FeoB. It will address the unknown transport of iron across the inner membrane, by showing if FeoB is responsible for direct transport of ferrous iron or not. This insight would be a novelty and could open doors to a variety of new research options.

1 . 9 . S i g n i f i c a n c e

The proposed duration for this project for a period of one year is not sufficient to find the answers to all the unanswered questions. Yet all additional information about the mechanism of iron binding and transport of FeoB will lead to a higher understanding of this essential process. Once the iron binding process is fully understood, FeoB could become a potential target for treatment. This would lead to many new research options and possibly to the development of new drugs targeting FeoB. It would be a major step forward in dealing with the rising problem of antibiotic resistance of pathogens, as FeoB and its homologs are commonly expressed in a majority of known bacteria (Carpenter and Payne, 2014).

Page 32 of 88

2 . M a t e r i a l a n d M e t h o d s

2 . 1 . M a t e r i a l s

All general reagents, biochemicals and equipment were purchased from Sigma-Aldrich unless otherwise stated (e.g. growth media, DDM, Ni-NTA, etc.

Including mant-ATP/-GTP and anti-his/anti-mouse antibodies). Purelink quick plasmid miniprep kit, DH5α E. coli cells, and iBlot dry blotting kit were purchased from Invitrogen. Crosslinker BMOE and TMEA were purchased from Thermo Fisher scientific. Precision Plus Dual Colour MW standard, Bio-spin disposable chromatography columns and Bio-Rad protein determination kit were purchased from Bio-Rad. Hyperladder I was purchased from Bioline.

Monobromobimane and dibromobimane were purchased from Molecular Probes.

2 . 2 . P l a s m i d p r e p a r a t i o n

Protein expression plasmids were propagated in E. coli DH5α cells in 5ml LB medium, from which plasmid DNA could be prepared using Purelink Quick Plasmid Miniprep Kit. Kit instructions were followed using O/N cultures of glycerol stocks of each DH5α strain to isolated plasmid DNA. Cells were harvested, lysed and non-plasmid DNA precipitated, resulting in pure plasmids in the pET-41a vector for each strain. Resultant samples were confirmed for purified plasmids using 1% agarose gel electrophoresis run with Hyperladder I.

C41 (DE3) overexpress E. coli cells from Lucigen.

2 . 3 . C o m p e t e n t c e l l s

An O/N culture of E. coli C41(DE3) was prepared from glycerol stocks in 5ml LB medium (1% Tryptone, 0.5% Yeast Extract. 1% NaCl) and incubated at

Page 33 of 88 37°C. From this O/N culture 0.5mL was used to inoculate 50mL SOB medium (2% tryptone, 0.5% yeast extract, 0.24% MgSO₄, 0.05% NaCl, 0.0186% KCl) containing 1% sterile glycine. Cells were grown at room temperature with slow shaking until an OD660 of 0.5-0.6 was reached at which point cells were rested on ice for 10 minutes before harvesting (1,400g, 4°C, 10 minutes). The supernatant was discarded and the pellet resuspended in 2ml of ice cold transformation buffer (10mM PIPES pH 6.7, 15mM CaCl2, 250mM KCl, 55mM MnCl2), to which 0.3mL DMSO was added before resting on ice for 10 minutes.

Competent cells were divided into 220µL aliquots in 1.5mL Eppendorf tubes which were then snap-frozen in dry ice/EtOH for storage at -80°C.

2 . 4 . T r a n s f o r m a t i o n

Competent cells C41 (DE3) were thawed on ice; at which point 1 µL pET41-a supercoiled plasmid (FeoB WT, D123N, C421S, C675S, respectively) was added to 100µL competent cells. The cells were then rested on ice for 30 minutes (as well as an empty vector transformation prepared for negative controls). Cells were heat shocked at 42°C for 45 seconds and then rested on ice for 2 minutes. Cells were grown by adding 100µL SOC (SOB containing 0.5% sterile glucose) which were then incubated at 37°C for 1 hour with slow shaking. 100µL of cells culture was plated on LB agar (1% Tryptone, 0.5%

Yeast Extract, 1% NaCl, 1.5% Technical Agar No. 3) containing 25µg/ml Kanamycin. To ensure a sufficient number of colony forming units were grown on the transformation plates the remaining cells were concentrated by spinning at 800g for 3 minutes and resuspended in the final 100µL of supernatant. This 100µL was plated on LB agar containing 25µg/mL kanamycin, and both LB agar plates were grown overnight at 37°C.

Page 34 of 88 2 . 5 . I n s i d e - O u t v e s i c l e p r e p a r a t i o n

ISOV were always prepared from freshly transformed cells. A pre-inoculation of 200mL LB medium, 25µg/mL kanamycin and 7-10 large colonies from transformation plates was prepared and incubated O/N at 37°C with slight shaking. This O/N culture was used to prepare a 2% inoculum in 8x800mL pre-warmed TB medium (1.2% tryptone, 2.4% yeast extract, 0.94% K2HPO4, 0.22% KH2PO4, 0.8% glycerol) containing 25µg/mL kanamycin. TB medium was grown at 18°C with shaking until cells reached an OD660 of approximately 0.3-0.5, at which point protein expression was induced with 0.5mM IPTG O/N. With the remaining procedure all completed on ice, cells were harvested by centrifugation (6,000g, 4°C, 15 minutes). FeoB cells were resuspended in approximately 200mL 100mM K-HEPES pH 7.0 to which 10µg/mL DNAse and 10mM MgSO4 were added and incubated at room temperature for 15 minutes to allow for DNA digestion. Cells were lysed by passing them 3x through Constant Systems Cell Disruptor and then incubated at room temperature for 15 minutes to allow for DNA digestion. Debris and unlysed cells were removed by centrifugation at (13,000g, 4°C, 10 minutes).

FeoB ISO vesicles were harvested from supernatant by centrifugation (150,000g, 4°C, 1 hour). Supernatant was removed and ISOV pellet was resuspended in approximately 8mL 50mM K-HEPES pH 7.0 containing 10%

glycerol. ISO vesicle samples were separated into 1mL aliquots and snap frozen in dry ice/EtOH for storage at -80°C.

2 . 6 . P r o t e i n p u r i f i c a t i o n

Protein purification was carried out on ice or at 4°C. Membrane proteins were solubilised for 2h at RT in solubilisation buffer (10mM K- HEPES pH 7.5, 20%

glycerol, 500mM NaCl, 2% DDM, 10mM imidazole) on Hoefer Red Rocker at highest speed (7-8) without bubbling of detergent. Unsolubilised particles

Page 35 of 88 were removed by centrifugation (150,000g, 4°C, 1 hour). Supernatant was bound to Ni-NTA resin of resin volume 100µl per 100mg total protein for a minimum of 1 hour at 4°C (before use resin was washed in 2x20ml water consecutively to remove ethanol). The Ni-NTA resin was transferred to a 2ml polystyrene mini-column and unbound protein fraction was drained and collected. The resin was washed as described by Seyedmohammad et al.

(2014), with 20 resin volumes wash buffer A (10mM K- HEPES [pH 7.0], 10%

glycerol, 10mM imidazole, 500mM NaCl, 0.05% DDM) followed by 20 resin volumes wash buffer B (10mM K- HEPES [pH 7.0], 10% glycerol, 10mM imidazole, 50mM NaCl, 0.05% C₁₂E₈). Five resin volumes of elution buffer (10mM K- HEPES [pH 7.0], 10% glycerol, 500mM imidazole, 200mM NaCl, 0.05% C₁₂E₈) were added to column. The first 0.5 resin volumes of drained solution were discarded and the following 3-5 resin volumes containing eluted protein were collected.

2 . 7 . P r o t e i n d e t e r m i n a t i o n

Purified protein concentrations were determined through use of Nanodrop 2000 Spectrophotometer, using elution buffer to blank the instrument. ISO vesicle protein concentrations were determined through the use of Bio-Rad protein determination kit with BSA as a standard. Dilutions of BSA (0- 1.5mg/mL) were prepared in water to create standard curve and 50-100x dilutions prepared for ISO vesicle samples. 20µL of all samples were pipetted into fresh Eppendorf tubes to which 100µL of reagent A was added and the tube vortexed. Reagent B (800µl) was added and the tubes vortexed immediately. Reactions were rested at room temperature for 15 minutes and the absorbance’s read at 750m, which allowed for the plotting of a standard curve (exemplary STD curve shown in Figure 6) and determination of protein concentration in unknown samples.

Page 36 of 88

Figure 6: Exemplary BSA standard curve. Absorbance of BSA in different concentrations (0-1.6mg/ml) was measured at OD750.

2 . 8 . S D S - P A G E

For detection of FeoB protein, samples were separated using 10% T solution running gel (10% acrylamide: bis solution (37.5:1), 375mM Tris pH 8.8, 0.1%

SDS, 0.05% APS, 15.3% TEMED) with a 5% T stacking gel (5% acrylamide:

bis solution, 250mM Tris pH 6.8, 0.1% SDS, 0.05% APS, 0.1% TEMED).

Approximately 30µg total protein (5µg for purified samples) was prepared in loading buffer 4x NuPAGE LDS sample buffer, with or without 3% β- mercaptoethanol, which was diluted to a final concentration of 1x with sample.

Sample with loading buffer was incubated at room temperature for 20 minutes followed by loading and running sample in gel at 80-100V in 1x SDS tank buffer (0.3% Trisma base, 1.876% glycine, 0.1% SDS). All gels were run with Bio-Rad Precision Plus Dual Colour standards. Gels were staining using Coomassie stain (10 % acetic acid, 40% methanol, 1% Coomassie Brilliant Blue R-250), destained in destaining solution (7% acetic acid, 5% methanol) and imaged using ImageQuant LAS 4000.

y = 0,2533x + 0,081

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5

0 0,5 1 1,5 2

OD 750 [nm]

Protein concentration [mg/ml]

BSA Standard

BSA Standard Linear (BSA Standard)

Page 37 of 88 2 . 9 . W e s t e r n b l o t

Protein detection was performed using Invitrogen iBlot Dry Blotting System and the iBlot Gel Transfer Stacks PVDF Mini. SDS-PAGE was run as per 2.6 SDS-PAGE, without staining/destaining. iBlot was prepared following providers instructions with Anode Stack at the bottom, gel placed on the transfer membrane of the Anode stack followed by a pre-soaked Filter paper (soaked in deionized water). Air bubbles were removed by using the blotting roller. Out of the Cathode stack top was the cathode taken and placed on the pre-soaked Filter paper with the electrode side facing up. At this point it was ensured all bubbles were removed by using the roller again. The disposable sponge was placed with the metal contact on the upper right corner of the lid and the lid was shut then. Transfer was achieved through blotting with Program 0. This program runs for 7 minutes in total and uses 20V for 1 minute, 23V for 4 minutes and 25V for the remaining 2 minutes. Following disassembly of apparatus, transfer was checked with Poncheau S solution, which is washed off with water after positive detection of protein on transfer membrane. To block free membrane protein binding sites, membrane was blocked with 25mL fresh TBST (50mM Tris pH 7.4, 150mM NaCl, 0.1% Tween20) containing 5% skim milk powder for 1 hour. Subsequently the membrane was incubated for 1 hour with TBST and 1/1000 anti-his antibodies. Unbound antibody solution was removed through washing of the membrane with TBST for 10 minutes, twice.

Membrane was incubated for 1 hour with 1/5000 anti-mouse antibodies.

Unbound antibody solution was removed by washing with TBST for 10 minutes, three times. For detection of antibody conjugation 2mL each of ECL reagents 1 and 2 was added and left for 5 minutes. Excess reagent was run off the membrane and the membrane wrapped in cling film. Membrane was imaged using ImageQuant LAS 4000 with 1 minute exposure for ISOV and 0.5 to 1 second exposure for pure protein blots.

Page 38 of 88 2 . 1 0 .C r o s s l i n k i n g w i t h d i b r o m o b i m a n e

Purified Protein was reduced by incubation with 5x molar excess of DTT for 20 minutes. 25µg of protein was then diluted in 200µl of assay buffer (10mM K-HEPES, 10% Glycerol, 200mM NaCl, 0.05% C12E8, 10mM MgSO4, 5mM Ascorbic Acid and filled up with MilliQ to 200µl). Half the protein samples and buffer were incubated with 1 mM GTP and 100µM FeSO4 for 20 minutes at RT under slight shaking. Pre-incubated and non-treated samples each were pipetted in dublicates onto a black 96 well plate with clear, flat bottom. The fluorescent probe bBBr was added in 10x molar excess to each well and the plate was immediately read with BioTek Cytation 5 imaging reader using the program Gen5 Image with continuous linear shaking, excitation wavelength of 393nm, emission wavelength of 475nm and the optics in top position. The fluorescence was measured every 10 minutes for a total of 3 hours.

2 . 1 1 .C r o s s l i n k i n g o f p u r i f i e d F e o B

Purified protein was prepared to a final concentration of 20µg in assay buffer (10mM K-HEPES, 200mM NaCl, 0.05% C12E8, 10mM MgSO4, 5mM Ascorbic Acid) a total volume of 30µl. Prior to crosslinking the samples were incubated with either 2mM GTP or 2mM non hydrolysable GTP-gamma-analogue, 100µM FeSO4 and 1mM Protease Inhibitor cocktail for 20 minutes under mild shaking at RT in order to open the pore. Either bifunctional (BMOE) or trifunctional (TMEA) cross linker was added in 5 times molar excess of protein and incubated at RT for 1 hour (bifunctional, BMOE) or 2 hours (trifunctional, TMEA). The crosslinking reaction was stopped by addition of 30µM DTT.

Following the crosslinking 5µg protein sample was mixed with 3µl deionised water and 3µl loading buffer and incubated for 20 minutes at RT. After incubation samples were loaded onto a 4-12% gradient SDS-gel (Invitrogen) and run for 40 minutes at 165V in Invitrogen MOPS running buffer. All gels