Part:BBa_K1639003

TlpB

Helicobacter Pylori TlpB protein coding region. This part gives a negative chemotactic response to H+ ions. This makes bacteria capable of moving from acidic pH to more neutral pH levels.

Usage and Biology

Chemotaxis, 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.

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).

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 the methylation 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.

Characterization

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:

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 Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2
  Illegal XhoI site found at 1710
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]