ULCER
Acid Resistance
The main goal of this part is; making the E. coli, which we used for eradicating H. Pylori, acid resistant for living in gastric juice’s low pH (pH: 2) microenvironment. Wild-type E. coli already has couple systems to show acid resistance until a certain pH level (pH: 5). Among those systems, the most stable-working and efficient one is known as Glutamate Dependent Acid Resistance(GDAR) system. The most important protein of Gad system is GadE, which controls the synthesis of all other proteins. By overexpressing this protein, we aim to have our bacteria resistant enough to live in gastric juice for a while.
BackgroundEscherchia coli Natural Acid Resistance System
E. coli possesses four phenotypically distinct systems of acid resistance. These systems:
1. Glutamate dependent acid resistance system (GDAR)
2. Arginine dependent acid resistance system (ADAR)
3. Lysine dependent acid resistance system (LDAR)
4. Ornithine dependent acid resistance system (ODAR)
The most effective one of these systems is the glutamate dependent system so we decided to focus on glutamate dependent system (GDAR).
The GDAR system has in the acid resistance complex pathways. We decided to do the certain pathways that how to do acid resistance with our results of research.
EvgA is the most effective positive regulator of GDAR. (Efficiency: evgA>ydeO>gadE) But evgA takes part in countless number different genes regulation and cellular processes (2,6,7) and also most of this cellular processes are unclear. We cannot predict the results of the overexpression of evgA so we chose gadE and using by overexpression of gadE we can induce glutamate depended acid resistance system.
Briefly talk about the impact of the GadE on the mechanism.
The central activator is the LuxR-family member GadE (formerly known as YhiE). GadE binds to a 20-bp sequence called the gad box, which is located 63-bp upstream of the transcriptional start sites of gadA and gadBC. (4)GadE and GadBox are an important point for GadA and GadBC. This means that GadE and GadBox are an important for acid resistance. The basis section of acid resistance is GadE’s regulation. At least 10 different regulatory take part in this regulation.
GadE have three main activation mechanisms. The first of these is performed by evgA and ydeO. About GadE activation phase:
1. EvgS (sensor kinase) activate EvgA (response regulator)
2. YdeO and evgA are independently from each other and GadE takes an active role in transcription activation.
The second GadE activation circuit includes CRP,RpoS and two AraC-like regulators, GadXand GadW.
These steps are as follows:
1. GadX and GadW, are located downstream of gadA also GadX and GadW directly activate transcription of gadE
2. GadX and GadW also bind to the gadA and gadBC gad box sequences and seem to repress the gadA and gadBC promoters. (4)
GadW inhibits this GadX’s repression
Also GadX and GadW regulate indirectly GadA/BC transcriptional functions.
3. The balance of power in this circuit is influenced by cAMP and CRP,which together inhibit the synthesis of RpoS.
Growth under acidic conditions reduces the concentration of cAMP in the cell.
RpoS increases GadX’s transcriptional function
The increase in GadX then stimulates transcription of GadE and also down regulates GadW.
The third activation of GadE contains TrmE and glucose. These steps are as follows:
1. The function of TrmE in the cell is not fully defined but it does have a clear effect on tRNA modification
2. TrmE and glucose increase independently GadE’s transcriptional function
We discourse GadE’s regulation and then now we will discourse GadE’s effect mechanisms.
GadA/BC’s effect mechanisms to acid resistance are as follows:
1. The external pH is normally neutral but if external pH turns to acidic pH, internal pH begins to become acidic pH with HCl diffusion.
2. If the external pH=2,5 and internal pH begins pH=4.2 ± 0.1. GDAR system will activate for acid resistance.
3. GadC is a transmembrane protein. The external pH begins to change from neutral pH to acidic pH, C-plug (is GadC’s subunit) will open. Then glutamate will take inside.
4. Glutamate in cells convert to GABA by GadB/C that’s glutamate decarboxylase isozymes
These steps are as follows:
*This step contains pyridoxal phosphate-containing enzymes that replace the α-carboxyl groups of their amino acid substrates with a proton that is recruited from the cytoplasm.
*HCl that diffusion from outside dissociates H+ and Cl-. This H+ is used and CO² is released by the agency of GadA/B isozymes than this is the last stage of glutamate changes to GABA. Besides this is the most important and the last step for acid resistance
5. If H+ leaves HCl it is staying back Cl and it will be export from the transport of chlorine channel.
6. The emitted CO² is taken out by diffusion.
7. Finally, the uncovered GABA is thrown out from the transmembrane protein GadC.
In this section, we targeted to overexpress GadE protein. For this purpose, while we’re desinging the gene part, we chose to combine GadE sequence with T7 promoter, a promoter which has a high rate of transcription. In addition to this, to have a controllable system, LacI protein attachable Lac operator was added between T7 and GadE gene sequence. We searched to find a vector which can lay down all those conditions and found out that peT-45 expression vector is useful. When we ordered our genes, we put RFC10 prefix site on 3’ end of GadE and also BamHI restriction enzyme recognition site for cloning to peT-45. We added RFC10 suffix site on 5’ end of the same gene sequence and also XhoI restriction enzyme recognition site for cloning to peT-45 vector, again.
As shown above,we planned to clone the ordered GadE gene into peT-45 vector by using BamHI and XhoI enzymes. Final construct after this cloning is, respectively T7 promoter-Lac operator(LacO)-HisTag-GadE. Besides, a constitutive promoter called LacI promoter and LacI protein sequence in front of that, found on peT45-b vector’s another part. It is obvious that this part is IPTG-dependent, so Western Blot can be performed easily with the help of His Tag.
GadE-PSB1C3 CLONNING
We cloned IDT G-Blocks GadE gene into PSB1C3 vector in order to make it ready to be submitted and have many copies of it. For this purpose we digested PSB1C3 vector and GadE G-Blocks with ECoRI and PstI restriction enzymes. Then we ligated these cut genes into the plasmid by using T4 DNA Ligase . Ocurring products were transformed into BL21 competent cell strain.
To check if the cloning is correct, a colony PCR was perfomed with Verify Forward and Verify Reverse primers. If the cloning isn’t made properly t the band should be 314 bp long, but if colony PCR worked, then bands should be 858 bp long.
As the result of colony PCR, the possible right cloned colonies were incubated in liquid culture for 16 hours. After this incubation, we isolated plasmid DNA from this bacteria culture by miniprep plasmid isolation method. We controlled obtained colonies with cut-check for a second cloning control. We used EcorI and PstI restriction enzymes for cut-check.
GadE-pET45-b CLONNING
We cloned GadE into PSB1C3 vector successfully, then moved on cloning it into the expression vector pET45-b. For cloning into this plasmid, we removed our gene from PSB1C3-GadE plasmid with BamHI and XhoI enzymes. Then we ligated the cut gene with pET45-b which was cut with the same enzymes. We transformed ligated products into BL21 bacteria strain that we know it has T7 RNA polymerase.
To check if the cloning is correct, a colony PCR was performed with T7 Promoter Forward and T7 Terminator Reverse primers. If the cloning isn’t made properly the band should be 360 bp long, but if colony PCR worked, then bands should be 837 bp long.
As the result of colony PCR, the possible right cloned colonies were incubated in liquid culture for 16 hours. After this incubation, we isolated plasmid DNA from this bacteria culture by miniprep plasmid isolation method. We controlled obtained colonies with cut-check for a second cloning control. We used BamHI and XhoI restriction enzymes for cut-check.
WESTERN BLOTTING
After cloning GadE into pET45-b successfully, we did Western Blot experiment through N-terminal located His Tag in proteins, so we managed to show the production of required proteins.
FUNCTIONAL ASSAY
To understand if the proteins we produced are functional, we designed and performed a functional assay. We incubated these two types of bacteria in liquid culture for 13 hours at 37 C; pET45-GadE plasmid containing BL21 bacteria and another BL21 bacteria which contains only pET45-b plasmid for negative control. At 13. our, we added 100 mM IPTG into these liquid cultures. By doing this, we removed the suppression on GadE protein expression. After adding IPTG, we incubated 3 hours more at 37 C.
We prepared LB mediums with different pH values to show produced GadE proteins’ functionality. These LB mediums’ pH values are respectively 7, 5, 3,5; 2,5 and 2. We added thebacteria whichis incubated for 16 hours into LB mediums at the rate of 1:9. This means, for each pH value we added 0.5 ml liquid culture into 4.5 ml LB medium. We also added 1.5 mM Glutamat in each mix and incubated the final mix at 37 C. We made spectrophotometric measurement in 600 nm periodically for the samples that we incubated. Thus we observed how long the bacteria survives in different pH values.
The measurement results are shown below.
1.5 mMGlutamate / 600 nm OD / 3h 100 mM IPTG
When the given data checked, it is obvious that the produced GadE is functional and makes E. coli survive in the acidic microenvironment in comparison to negative control results. Based on the data given, the E. coli with overexpressed GadE can survive in pH 2 gastric juice for 5-9 hours.
Acid Repellency
The main objective of this part of our project is to direct resistant E. Coli into the internal mucus layer where Helicobacter Pylori are abundant for a length of time. We will direct our E. Coli using the hydrogen ion difference between the stomach fluid and mucus layer. The PH of gastric juice is 2.0 whereas the pH of gastric mucus layer is 7.0. The chemoreceptor tlpb naturally found in the inner membrane of helicobacter pylori enable these bacteria to move away from acidic condition to a more neutral and basic environment. Therefore, we decided to use the TIpB protein and designed a biobrick for our E. Coli in order to ensure that our Bacteria reach the region where the helicobacter pylori are.
BackgroundChemotaxis, movement toward or away from chemicals, is a universal attribute of motile cells and organisms. E. coli cells swim toward amino acids (serine and aspartic acid), sugars (maltose, ribose, galactose, glucose), dipeptides, pyrimidines and electron acceptors (oxygen, nitrate, fumarate).
E. coli's optimal foraging strategy
In isotropic chemical environments, E. coli swims in a random walk pattern produced by alternating episodes of counter-clockwise (CCW) and clockwise (CW) flagellar rotation (Fig. 3, left panel). In an attractant or repellent gradient, the cells monitor chemoeffector concentration changes as they move about and use that information to modulate the probability of the next tumbling event (Fig. 3, right panel. These locomotor responses extend runs that take the cells in favorable directions (toward attractants and away from repellents), resulting in net movement toward preferred environments. Brownian motion and spontaneous tumbling episodes frequently knock the cells off course, so they must constantly assess their direction of travel with respect to the chemical gradient.
Figure 1: Random and biased walks. Left: A random walk in isotropic environments. When the cell's motors rotate CCW, the flagellar filaments form a trailing bundle that pushes the cell forward. When one or more of the flagellar motors reverses to CW rotation, that filament undergoes a shape change (owing to the torque reversal) that disrupts the bundle. Until all motors once again turn in the CCW direction, the filaments act independently to push and pull the cell in a chaotic tumbling motion. Tumbling episodes enable the cell to try new, randomly-determined swimming directions. Right A biased walk In a chemoeffector gradient. Sensory information suppresses tumbling whenever the cell happens to head in a favorable direction. The cells cannot head directly up-gradient because they are frequently knocked off course by Brownian motion..
The chemotaxis signaling pathway of E.coli
E. coli senses chemoeffector gradients in temporal fashion by comparing current concentrations to those encountered over the past few seconds of travel. E. coli has four transmembrane chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), that have periplasmic ligand binding sites and conserved cytoplasmic signaling domains (Fig. 4). MCPs record the cell's recent chemical past in the form of reversible methylation of specific glutamic acid residues in the cytoplasmic signaling domain (open and filled circles in Fig. 4). Whenever current ligand occupancy state fails to coincide with the methylation record, the MCP initiates a motor control response and a feedback circuit that updates the methylation record to achieve sensory adaptation and cessation of the motor response. A fifth MCP-like protein, Aer, mediates aerotactic responses by monitoring redox changes in the electron transport chain. Aer undergoes sensory adaptation through a poorly-understood, methylation-independent mechanism. The five MCP-family receptors in E. coli utilize a common set of cytoplasmic signaling proteins to control flagellar rotation and sensory adaptation (Fig. 4). CheW and CheA generate receptor signals; CheY and CheZ control motor responses; CheR and CheB regulate MCP methylation state.
Figure 2: Signaling components and circuit logic. E. coli receptors employ a common set of cytoplasmic signaling proteins: CheW and CheA interact with receptor molecules to form stable ternary complexes that generate stimulus signals; CheY transmits those signals to the flagellar motors, CheZ controls their lifetime; CheR (methyltransferase) and CheB (methylesterase) regulate MCP methylation state. Abbreviations: OM (outer membrane); PG (peptidoglycan layer of the cell wall); CM (cytoplasmic membrane)
As in many biological signaling systems, the signaling currency in the E. coli chemotaxis pathway is reversible protein phosphorylation (Fig. 5). However, the principal signaling chemistry is a bit different in prokaryotes and eukaryotes. CheA is a kinase that uses ATP to autophosphorylate at a specific histidine residue. Phospho-CheA molecules then serve as donors for autokinase reactions that transfer phosphoryl groups to specific aspartate residues in CheY and CheB. Phospho-CheY enhances CW rotation of the flagellar motors; phospho-CheB has high MCP methylesterase activity. The active forms of these response regulators are short-lived because they quickly lose their phosphoryl group through spontaneous self-hydrolysis. CheZ further enhances the dephosphorylation rate of phospho-CheY to ensure rapid locomotor responses to changes in the supply of signaling phosphoryl groups to CheY.
CheW couples the autophosphorylation activity of CheA molecules to chemoreceptor control. Receptors, CheW, and CheA form stable ternary signaling complexes that modulate the influx of phosphoryl groups to the CheY and CheB proteins in response to chemoeffector stimuli.
Figure 3: Phosphorelay signaling. The flagellar motors of E. coli spin CCW by default; the signaling pathway modulates the level of phospho-CheY, the signal for CW rotation. Reactions and components that augment CW rotation are depicted in green; those that augment CCW rotation are depicted in red.
Chemoreceptor signaling states in E.coli
The signaling activities of chemoreceptors are described by a two-state model (Fig. 6). Receptor complexes in the CW signaling state activate CheA, producing high levels of phospho-CheY. Receptors in the CCW signaling state deactivate CheA, resulting in low levels of phospho-CheY. Thus, the behavior of the flagellar motors reflects the relative proportion of receptor signaling complexes in the kinase-on and kinase-off conformations. Both chemoeffector binding or release and methylation or demethylation can shift receptor signaling complexes from one state to the other. For example, attractant ligands drive receptors toward the kinase-off state; subsequent addition of methyl groups shifts receptors toward the kinase-on state, reestablishing the steady-state (adapted) balance between the two states and restoring random walk movements.TlpB in Helicobacter Pylori:
H. pylori is a Gram-negative bacterium that resides in thestomachs of over half the world’s population. Its gastric habitatcontains a marked pH gradient from the highly acidic lumen,which can reach pH 2, to the more neutral environment adjacentto the epithelial lining, which is typically pH 7. Based on genome sequence analysis, the H. pylori chemotaxismachinery resembles that of the well-studied model, E. coli,with a few notable variations including the absence of themethylation enzymes involved in receptor adaptation (Sweeneyand Guillemin, 2011).
Figure 5: Chematic representation of chemotaxis signaling in E. coli (A) and H. pylori (B). Chemoreceptors are shown in purple and flagellar motors in greenspanning the cell membrane. Cytoplasmic chemotaxis proteins CheA, CheW, CheY, CheZ, CheR, CheB, CheV, and ChePep are labeled. Protein modificationsare shown as pink circles for phosphorylation and purple hexagons for methylation. Activating interactions between signaling pathway components are indicatedby arrows, and speculative interactions are indicated by dotted lines.
When Helicobacter pylori and Escherichia coli bacteria which belong to two separate systems are examined, we recognize the vast similarity between the two. Both systems have chemoreceptors which belong to the MCP family enable the detection of chemical substances which do chemotaxis. Also, both bacteria also share in common the proteins required to send signals to necessary regions.
H. pylori has four chemoreceptors, TlpA,TlpB, TlpC, and TlpD, and several polar flagella that dictatesmooth swimming behavior in the presence of an attractantand increased stopping behavior in the presence of a repellent(Lowenthal et al., 2009; Rader et al., 2011). Only a small numberof chemotactic signals have been identified for H. pylori. Thebest-haracterized chemoreceptor is TlpB, which is requiredfor chemorepulsive responses to acid, as well as the quorumsensingmolecule autoinducer-2 (AI-2) and H+. TlpB is the first bacterial chemoreceptor of known function shown by crystallography to contain an extracellular PAS domain. Strikingly, a molecule of urea is found within the canonical ligand-binding site of the PAS domain, bound in a manner predicted to be sensitive to changes in pH.
Figure 6: TlpB Forms a Dimer that ContainsUrea-Binding PAS Domains(A) Schematic of estimated TlpB domains is illustrated.Transmembrane region (TM, orange);PAS domain (light gray); HAMP domain (green);chemoreceptor trimer of dimers contact region(blue). The periplasmic domain is from aminoacids 32–209.(B) Ribbon diagram of TlpBpp homodimer andgray/blue/red/white urea molecules, with chaincolor gradation ranging from N terminus (blue) to Cterminus (green) for the monomer on the right andN terminus (light green) to C terminus (red) for themonomer on the left is presented. For orientationthe bacterial inner membrane would be below thelower part of the protein model diagram.(C) Diagram of the TlpBpp urea-binding siteincluding hydrogen bonds (dashed lines) betweenurea (gray, white, blue, and red) and the surroundingresidues and water molecule is demonstrated.Oxygen atoms are shown as red spheres,nitrogen as blue, and hydrogen as white..
Emily Goers Sweeney et.al. report that urea is bound with extremely high affinity andspecificity and is essential for the thermodynamic stability ofthe TlpB molecule. The urea-binding site includes an aspartategroup (Asp114), which we propose to be the key titratableresidue responsible for pH sensing. Mutational and biophysicalanalyses of the urea-binding site support a mechanism in whichthe urea cofactor, by binding in a pH-sensitive fashion, stabilizesthe secondary structure of TlpB and signals a pH response.
Figure 7: Model for how TlpB Senses Acid: In low pH conditions (shown on the left), TlpB’s periplasmic domain is ina ‘‘relaxed’’ or expanded state due to decreased hydrogen bonding to ureaand consequent lowered urea-binding affinity. However, in high or moreneutral pH conditions, TlpB’s periplasmic domain is in a ‘‘tense’’ or condensedstate with increased urea-binding affinity. The state of the periplasmic domainis relayed through the transmembrane region, which affects CheA’s phosphorylationstate, ultimately affecting the flagellar motor and dictating stoppingbehavior. TlpB dimer is shown in orange, CheA in red, CheW in green, urea inpurple, protons in blue, and phosphate in black..
By sequence analysis, H. pylori TlpB is organized like a typical member of the MCP superfamily of transmembrane receptors with two transmembrane helices (tm1, tm2) per subunit bracketing an extracellularsensing domain[2,7]. The extracellular-sensing domain is responsible for detecting ligands directly or indirectly via interactions with periplasmic-binding proteins. Continuing from tm2, the C-terminal portion of the MCP is cytoplasmic and mostly helical. It contains a histidine kinase, adenylyl cyclase, methyl-binding protein, phosphatase (HAMP) domain, followed by a helical domain and a segment that binds to the CheA/CheW histidine autoki-nase complex[2,8]. Phosphorylation of CheA in response to an extracellular signal in turn controls downstream compo-nents that modulate activity of the flagellar motor. MCPs dimerize at the membrane, forming a four helix bundle with ligand-binding domains in the periplasm, whereas trimers of dimers assemble with CheA and CheW to form a high-perfor-mance signaling array located in the cytoplasm[2,9].
In low pH conditions , TlpB’s periplasmic domain is in a ‘‘relaxed’’ or expanded state due to decreased hydrogen bonding to urea and consequent lowered urea-binding affinity. However, in high or more neutral pH conditions, TlpB’s periplasmic domain is in a ‘‘tense’’ or condensed state with increased urea-binding affinity. The state of the periplasmic domain is relayed through the transmembrane region, which affects CheA’s phosphorylation state, ultimately affecting the flagellar motor anddictating stopping behavior
References
[1]Sachs, G., Weeks, D.L., Melchers, K., and Scott, D.R. (2003). The gastric biology of Helicobacter pylori. Annu. Rev. Physiol. 65, 349–369.
[2]Structure and Proposed Mechanism for the pH-Sensing Helicobacter pylori Chemoreceptor TlpB Emily Goers Sweeney,J. Nathan Henderson, John Goers, Christopher Wreden, Kevin G. Hicks, Jeneva K. Foster,Raghuveer Parthasarathy, S. James Remington,and Karen Guillemin
[3]Sweeney, E.G., and Guillemin, K. (2011). A gastric pathogen moves chemo-taxis in a new direction. MBio 2, e00201-11.
[4]Lowenthal, A.C., Simon, C., Fair, A.S., Mehmood, K., Terry, K., Anastasia, S., and Ottemann, K.M. (2009). A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility.Microbiology 155, 1181–1191.
[5]Croxen, M.A., Sisson, G., Melano, R., and Hoffman, P.S. (2006). The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa. J. Bacteriol. 188, 2656–2665.
[6]Hazelbauer, G.L., Falke, J.J., and Parkinson, J.S. (2008). Bacterial chemore-ceptors: high-performance signaling in networked arrays. Trends Biochem. Sci. 33, 9–19.
[7]Wuichet, K., Alexander, R.P., and Zhulin, I.B. (2007). Comparative genomic and protein sequence analyses of a complex system controlling bacterial chemotaxis. Methods Enzymol. 422, 1–31.
[8]Marchler-Bauer, A., Lu, S., Anderson, J.B., Chitsaz, F., Derbyshire, M.K., DeWeese-Scott, C., Fong, J.H., Geer, L.Y., Geer, R.C., Gonzales, N.R., et al. (2011). CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 39 (Database issue), D225–D229.
[9]Briegel, A., Ortega, D.R., Tocheva, E.I., Wuichet, K., Li, Z., Chen, S., Mu¨ller, A., Iancu, C.V., Murphy, G.E., Dobro, M.J., et al. (2009). Universal architecture of bacterial chemoreceptor arrays. Proc. Natl. Acad. Sci. USA 106, 17181– 17186.
[10]http://chemotaxis.biology.utah.edu/Parkinson_Lab/projects/ecolichemotaxis/ecolichemotaxis.html
In this section, our aim is synthesize the TlpB protein in our E.coli for to E.coli can penetrate to the stomach mucus layer, which is our pathogenic bacteria, H.pylori, resides.
For enhance the production of our protein TIpB, we aim to assemble our TIpB gene with T7 promoter which is a fast and strong promoter during we design our parts.
For controlling the system, we decided to add a Lac Operator, which is LacI protein can bind, between TIpB gene sequence and promoter. We searched for a expression vector that include Lac Operon and we found that peT45-b has the properties we want and we decided to use that vector.
When we order our genes we added RFC10 prefix site to 3’ end and RFC10 suffix site to the 5’ end of TlpB gene and also we added BamHI and XhoI restriction site sequence on the Pet45-b for cloning.
We planed to cloning TlpB gene that we ordered as described above to the Pet45-b vector with using BamHI and XhoI enzymes.
When this cloning achieved succesfully, there will be a construct that with order T7 Promoter-Lac Operon (LacO)- HisTag-TlpB and also there are LacI constructive promoter and LacI protein sequence ,in front of promoter, on the another area of Pet45-b vector . When these informations considered, it is clear that production of TIpB protein is IPTG depented and Western-Blot can be performed because of His-Tag site.
PSB1C3-TlpB CLONING
First of all we planned to cloning our gene that we ordered from IDT as a G-blocks to the PSB1C3 plasmid for multiply our genes and also for submitting to the iGEM as a biobrick in PSB1C3, for this purpose we cut both our PSB1C3 vector and G-Block of TIpB with EcoRI and PstI restriction enzymes. Then we inserted these G-Blocks to the plasmid with used T4 DNA ligase enzyme and we transformed our plasmid to the BL21 competent bacteria.
For verifying our cloning process we did colony PCR with Verify Forward and Verify Reverse primers. If we got the cloning results wrongly we should see 314 bp band but if we achieved the cloning we should see band.
After Colony PCR, for the clonings that we thought positive we put our bacteria to the 16 hours liquid culture and we isolated plasmid DNA with miniprep isolation method. We cutchecked our plasmid DNAs that we got from our isolation for controlling of cloning second time with using EcoRI restriction enzyme.
pET45-TlpB CLONING
After we cloned our genes to PSB1C3 plasmid successfully then we planned to cloning our genes to an expression vector pET45-b for high amount of protein synthesizing. For cloning our gene to this vector, we extracted our genes from PSB1C3-TIpB plasmid with using BamHI and XhoI enzymes. We performed ligation with pET45-b vector which is cutted with the same enzymes, then we transformed our plasmid to the BL21 competent bacteria which has a T7 RNA polymerase.
For verifying our cloning process we did colony PCR with T7 Promoter Forward and T7 Terminator Reverse primers. If we got the cloning results wrongly we should see 360 bp band but if we achieved the cloning we should see 2007 band.
After Colony PCR, for the clonings that we thought positive we put our bacteria to the 16 hours liquid culture and we isolated plasmid DNA with miniprep isolation method. We cutchecked our plasmid DNAs that we got from our isolation for controlling of cloning second time with using BamHI and XhoI restriction enzymes.
WESTERN BLOTTING
After we cloned our genes to pET-45b vector successfully then we did Western-Bloting for showing our proteins are synthesized with using the His-tag on the N-terminal of our proteins
SOFT AGAR PLUG ASSAY
For understanding that our proteins that produced are functional or not, we performed a chemotaxis motility assay which is called Agar Plug Assay.
We chose 2 different points on the soft agars that have 2 cm distance between them.We put the filter paper that is immersed in specially prepared liquid culture on the first point and then we put another filter paper that immersed in 0.2 mM HCL solution on the second point. Then we incubated our bacteria on 30C and examined their growing process on plates
The results are below
Increased Motility
In this section, the basic goal of the parts that we are using is to ensure that E. Coli can move from the acidic pH of the stomach fluid into the basic pH of the thick mucus layer and be able to move around within this area with ease. In this manner, the bacteria will have reached the region of the stomach where Helicobacter pylori is found. . Unlike normal fluids, the mucus layer is very thick and the flagella of natural E. Coli does not have sufficient torque or speed to allow it to enter.. In this project, we aim to give E. Coli the ability to enter the stomach’s mucus layer by increasing both of these characteristics. In order to achieve this goal, we plan to use 2 different strategies. . First, we will use a knock-out mutant form (HNS T108I) of the NHS transcription factor found in E. Coli. This mutant form has been shown to have a positive effect on the speed and torque of the flagella. The difference between the normal NHS and mutant NHS is a single amino acid change from threonine to isoleucine at residue 108 of the protein.
As for the second strategy to reach this goal, we aim to increase the torque power and flagellar speed by replacing the MotA and MotB proteins which make up E. Coli’s rotor (they form a H+ channel) with proteins PotB59 and PomA from V. Alginolyticus(they form a Na+ channel).
BackgroundHNS/HNST108I
Many species of bacteria have flagellar motility that is achieved by rotating surface-exposed organelles The rotation of the flagella is controlled by a motor complex embedded in the inner membrane (reviewed in Refs. 2 and 3 The motor region of E. Coli’s flagella is composed of several parts. The stator complex, made up of the proteins MotA-MotB, has a transmembrane characteristic and ensures the generation of a proton channel (4-6). The rotor complex involves the interaction of three other proteins, FliG, FliM, and FliN(7,8). All three rotor proteins are involved in the processes of flagellar assembly, switching, and rotation (9,10). However, FliG is predominately involved in torque generation (8, 10, 11), whereas FliM mainly functions in switching rotor direction (12). The precise role of FliN is the least well defined, but it may participate in flagella protein-specific export and assembly (13,14)
HNS is a transcription factor that plays a role in the regulation of gene expression in E. Coli. It plays an activator role at the level of protein synthesis for many genes involved in bacterial motility. . . In many studies it was shown that HNS is essential for the synthesis of proteins that make up the rotor and stator. The flhCD operon is a positive regulator of HNS which is responsible for the synthesis of many proteins that composes the structure of the bacterial flagella. Studies show that knock-out of the gene that codes the HNS protein results in non-motile E. Coli.
Above, we mentioned three proteins that make up the stator portion of the bacterial flagella.. These proteins are FliG, FliM and FliN and these three proteins have different activities. FliM is a regulator protein that is responsible for determining in which direction the bacterial flagella will be oriented. Although the role of FliN is not precisely known, it is thought that it acts as a guide for the assembly of the flagella’s membrane/for attachment of flagella to the membrane. As for FliG, this protein is responsible for the rotation speed and torque power of the bacterial flagella. For our aims in this part of the project, which are to increase the speed and power of the bacterial flagella, we decided to focus on FliG.
In a study by Gina M. Donato and Thomas H. Kawula, it was shown that HNS is not only a transcription factor but also that it can change some protein’s activities through direct interactions with them. . In studies including fluorescence anistropy and chemical cross-linking, it was shown that there is a physical interaction between HNS and FliG proteins.
Figure 2: Fluorescence anisotropy of H-NS-FliG interactions. Increasing amounts of FliG were added to fluorescein-labeled H-NS. Ten anisotropy values were measured at each FliG concentration and averaged. Graph is representative of two separate experiments. .
Figure 3: H-NS-FliG cross-linked complexes. Reactions were incubated at room temperature and equal amounts were electrophoresed on denaturing poly acrylamide gels. A, Coomassie-stained 4–20%SDSpolyacrylamidegradient gel; B, 12% SDS-polyacrylamide gel transferred to nitrocellulose, and probed with H-NS antiserum; C, second half of gel in B probed with FliG antiserum. Protein. Standard sizes are indicated by lines; lmw, low molecular weight markers; hmw, high molecular weight markers; protein monomers, dimers, and H-NS-FliG complexes are indicated by arrows. Reactions for all panels: 1, wild-type H-NS only; 2, H-NST108I only; 3, FliG only; 4, wild-type H-NS with cross-linker; 5, H-NST108I with cross-linker; 6, FliG with cross-linker;7, 1:1 molar ratio of wild-type H-NS to FliG with cross-linker; 8, 1:1molar ratio of H-NST108I to FliG with cross-linker.
Donato and Kawulainvestigated the effect on bacterial motility of random point mutations in the HNS protein. . As a result of their Swarm plate assay and experiments for the determination of flagellar rotational speed, it was found that some random mutations (HSN-T108I and HNS-A18E) resulted in bacteria with a stronger and faster flagella as compared with wild type bacteria. This single amino acid change caused a 50% increase in the binding of HNS to FliG.. It was also shown that this single amino acid change in HNS resulted in an approximately 50% increase in flagellar rotation speed and about a 2-fold increase in the bacteria’s swarm size.
Figure 4: Swarm plate assay. Fresh colonies from strains carrying the indicated alleles were inoculated onto semi-solid agar plates and grown at 30 °C. Growth was measured as the diameter of the bacterial swarm over several time points. Bacteria with swarm diameters under 10 mm at the end of 17 h incubation were considered non-motile. Data representative of three individual experiments. *, vector; f, hns2-tetR; ,,wild-type HNS; l, hnsT108I; l, hnsA18E..
Figure 5: Flagella propel bacteria by rotating motor-driven helical filaments (35, 36) whereby swimming speed is directly related to flagellar rotational speed (39). Gina M. Donato and Thomas H. Kawula1 , in another experiment which they tried to compare rotational speeds of mutant and wild-type bacteria, they concluded that hnsA18E and hnsT108I accelerated flagellar speeds 44–62% over wild-type levels.
In the results from fluorescence anisotropy and chemical cross-linking, Donato and Kawula showed that there is an interaction between HNS and FliG, the protein responsible for flagellar torque; and they show that the mutant form of HNS binds FliG 50% more often than does wild-type HNS: They explain the increase in the bacteria’s swarm rate and swim speed caused by mutant HNS thusly “We position H-NS at the interface between the rotor and stator, directly linked to the C terminus of FliG(Fig. 5 A). Tighter binding of mutant H-NST108I toFliG (Fig.5 B) may cause increases in flagellar speed by altering the conformation of FliG relative to the other rotor proteins and/or the MotA·B complex, thus, compacting the motor complex and allowing fast errotation by creating less friction within the surrounding stationary MotA·B ring complex (56).
This study having captured our attention, we decided to use mutant HNS to increase flagellar speed and torque power.
PotB59/PomA
The flagella of bacteria is made up of 30 different proteins and at least three different parts. These parts are the basal body, hook and filament. . The basal body is made up of the rotor and stator and is the region responsible for the rotation of the flagella. The stator is the part made up of channel proteins that generate the necessary proton motive force. The rotor is a stable region of the basal body which controls things such as the speed and direction of the flagella and is found in the inner region of the stator. The interaction between the rotor and stator, found in the basal body, result in the movement of the flagella. The filament is the long string-like structure that reaches outwards. . The role of the hook is to connect the filament and the basal body together.
Figure 1. Schematic diagram of the bacterial flagellum. The flagellum is a locomotive organelle for bacterial propulsion. The flagellum consists of the basal body, which acts as a reversible rotary motor, the hook, which functions as a universal joint and the filament, which works as a helical screw. OM, outer membrane; PG, peptidoglycan layer; CM, cytoplasmic membrane. .
The driving force of the bacterial flagellar rotation is an ion concentration gradient between the two sides of the membrane. The energy needed to rotate the flagella is generated by the majority of bacteria as a H or Na ion concentration gradient. In proton(H)-driven motors, the rotation of the flagella is carried out by the MotA and MotB membrane proteins. These proteins are actually H+ channels and when H+ moves through these channels depending on the concentration gradient, the necessary energy for flagellal rotation is created. This system that uses H+ is used by E. Coli and many other bacterial strains to generate energy for flagellar rotation. Other bacterial strains rely on Na+ to power their flagellar motor. These include Vibrio spp. (V. alginolyticus, V. parahaemolyticus ve V. cholerae) and alkaphilic Bacillus species. These bacteria, in place of MotA and MotB proteins, use homologous membrane proteins PomA and PomB. These proteins function as Na+ channels and ensure that the necessary energy for flagellar rotation is generated as Na+ passes along the channel. Furthermore, in bacteria that use Na+ channels to power their flagella, there are MotX and MotY proteins which are localized in the outer membrane These two proteins stabilize the binding of PomA/B proteins to the motor Flagella that work by Na+ have greater speed and rotational power that those that depend on H+.
Figure 2. Schematic diagram of the proton-driven bacterial flagellar motor. The flagellar motor consists of a reversible rotor made of FliF, FliG, FliM and FliN and a dozen stators, each of which consists of MotA and MotB. FliF forms the MS ring within the cytoplasmic membrane. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring. OM, outer membrane; PG, peptidoglycan layer; CM, cytoplasmic membrane.
Both MotA and PomA proteins have 4 membrane-spanning region, a huge cytoplasmic fold and a C-terminal end. Yet MotB and PomB has only one membrane-spanning region.
Figure 3. Arrangement of transmembrane segments of the MotA/B complex, which consists of four copies of MotA and two copies of MotB. The view is from the periplasmic side of the membrane. The complex has two proton conducting pathway shown by orange ellipsoids.
In work by M. Homma et al, a host of chimeric proteins were generated by combining different parts of the stator proteins (MotB and PomB) from the two systems mentioned above. . Many experiments were done to show the activity of the chimeric stator proteins and to identify the most effective of these. According to their data, the most effective protein was (PotB59)(PomB7E) which was generated by combining the N-terminal tail of PomB with the periplasmic C-terminal tail of MotB. . In order to understand how effective this protein worked, many experiments were conducted with this chimeric protein in combination with MotA and PomA. According to results from these experiments, the most effective rotor system was PomA/PotB59. This stator system Works with Na When this two protein system was established in E. Coli, it showed increased flagellar rotation speed and strength as compared to the original MotA/MotB stator system.
Figure 4. Amino acid alignments of V. alginolyticusPomB (Va), R. sphaeroides MotB (Rs), and E. coli MotB (Ec), and location of hypermotile mutations and junction sites of chimeric proteins. White letters in black boxes and arrows show identical residues and junction sites of chimeric proteins, respectively. Gray and hatched bars indicate putative transmembrane (TM) regions and peptidoglycan binding motif (PGB), respectively. Hypermotile mutations and strain numbers are indicated under the alignment.
Figure 5. Diagram of the properties of each chimeric B subunit. White, gray and hatched bars show the contributions of PomB or MotB from V. alginolyticus, R. sphaeroides and E. coli, respectively. TM, transmembrane region; PGB, peptidoglycan-binding motif. The stator proteins were expressed in V. alginolyticus or E. coli. R. sphaeroides MotA/MotB do not function in E. coli or V. alginolyticus, and PomA/PomB function only in V. alginolyticus. MotBE and PotB7E function in E. coli and V. alginolyticus. The properties of motors containing either protein are essentially the same in both species. Stators containing MomB7E or MomB7R function only in V. alginolyticus.
It is possible that the PomA/PotB59 system could be effective without the PotX and PotY proteins.
In light of the above information, we decided to use the PomA/PotB59 system in addition to the mutant HNS protein to increase the bacteria’s flagellar speed and torque strength.
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