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Ralstonia solanacearum

Ralstonia solanacearum belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as hrp (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. The secretion system is expressed only when there is a physical interaction between R. solanacearum and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, R. solanacearum binds to the cell surface. This union is independent of PrhA or any protein encoded by hrp. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the hrp regulatory gene. Induction of hrp expression is very fast, around 90 minutes, a period much shorter than generation time of R. solanacearum in optimal conditions. PrhA fundamental feature is that it is the first known bacterial receptor that can detect a non-diffusible signal present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of hrp, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.

Signal Transduction Circuits

Prh system of Ralstonia solanacearum

The Prh system integrates the genes involved in the control of expression of hrp virulence genes from Ralstonia solanacearum. hrp gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA PrhR PrhI proteins and other regulators that, as a last resort, activate the expression of the hrp or hrc (conserved hrp genes) genes.

The induction of virulence genes occurs when PrhA contacts with the plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction.

The prhIR gene expression is induced in coculture with plant cells for unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of PrhJ gene to initiate transcription. PrhJ protein induces hrpG transcription, which activates expression of hrpB gene and finally expresses hrp and hrc virulence genes.

The PrhA-PrhR-PrhI module of Ralstonia works similarly to FecA-FecR-FecI module of E.coli, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). The three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. The periplasmic N-terminal extension of FecA that is required for transduction of signals FecR, also appears in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecII are present in PrhR. However, unlike most of the siderophores, both prhIR and prhA lack Fur-boxes which are necessary for the regulation in function of the internal iron status.

Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, prhA constitutes a monocistronic operon at the left edge of hrp gene cluster and prhIR is on the right side of cluster, both prhA and prhIR separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed a very low level in the presence of the inducing signal and is PrhI independent.

To ensure that our system is able to detect the walls of plant cells and causes the excretion of chemoattractant, in order to be able to achieve effective bacterial accumulation around plant cells, we have designed four different circuits. These attempts to prevent the different problems that arise to us. The first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of R. solanacearum, but they are transfered to E. coli. The genes required for synthesis and excretion of the chemoattractant are under promoter PprhJ.

Because the Prh system is not fully characterized, unknown elements could be involved in and prevent the signal being correctly transmitted to PprhJ. For that reason, we have designed other circuits avoid this signaling cascade.

Fec system of Escherichia coli

Fec system includes genes involved in regulation and expression of E. coli iron transporters. fecABCDE genes express the ferric citrate transporter when bacteria iron status is low or deficient. The induction of genes fecABCDE integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module FecA-FecR-FecI is known as a signal transduction system between three compartments (outer membrane, periplasm and cytoplasmic membrane). The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of fecABCDE transport genes to initiate transcription. In addition, the transcription of regulatory genes fecIR is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2 +, it represses fecIR transcription and prevents the fec gene expression. Therefore, the fec transport gene transcription is subject to a double control: first, the cells detect iron deficiency. Then , regulatory proteins FecI and FecR are synthetised, which, if ferric citrate is available, initiate the transcription of fec transport genes.

Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.

In our second circuit, the iron transport genes (fecABCDE) are replaced for genes required for synthesis and excretion of the chemoattractant. Those genes remain under control of the promoter PfecABCDE, being regulated by the module FecA-FecI-FecR which is dependent of internal iron status and external ferric citrate concentration. This circuit has the advantage that, besides being well characterized, presents the components specific to E. coli. However, our system will not be directed specifically to plant tissues and would be regulated by iron status of the bacteria and the environment.

(FecA/PrhA)-FecI-FecR hybrid protein system

Our third circuit uses the FecA and PrhA sequence homology. We have designed a hybrid protein that detects the plant cell ligand and is capable of transmitting the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA. N-terminal is where FecA transmits the signal of FecR. The binding of both proteins was carried out by a shared sequence near Ton-box.

PrhA-fecI-FecR hybrid system

Due to the similarities between these proteins (FecA and PrhA), PrhA could interact with FecR by the end of it. However, this construction is largely a test, since the FecA sequence described that communicates with FecR is different in both proteins (FecA and PrhA).

Outer Membrane Protein Structures

Now it is going to be shown the structure of the outer membrane proteins which start the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.

Fe(3+) dicitrate transport protein fecA

FecA is the outer membrane receptor protein in the Fe dicitrate transport system of Escherichia coli. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:


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