Team:UniSalento Lecce/Overview

1 - Background

Nickel is a very widespread heavy metal and its levels in waters and other environmental matrixes are highly related to human activities. A lot of industrial applications leads to an increase of nickel amounts in the environment, and the growing nickel contamination led to an increase in Nickel related allergies (more than the 15% of the population suffers from nickel allergy) and other pathologies concerning the reproductive, cardiac and respiratory systems. Furthermore, Nickel compounds have been classified as carcinogenic by the International Agency for Research on Cancer, IARC.
The risks for human health made it necessary, as for many heavy metals, to remove this metal from the environment. Nowadays nickel can be chemically removed from fluids using a chelating agent, known as H2DMG (dimethylglyoxime),which helps the precipitation of the nickel metal ions for its dosing and removal.
We thought of a living organism carrying a nickel sensing device, coupled via Quorum Sensing to a second system (i.e. another bacterial population) which will remove the nickel from the environment. Our two-population system could be implemented in a water purification plant, allowing a regulated heavy metal removal without the need of chemicals (see the Applications page).

2 - Nickel sensing

The sensing device is based on a set of genetic parts from the pathogen Helicobacter pylori. This microorganism, which colonises the stomach, requires a large amount of nickel to maintain its homeostasis in such a hard environment. Particularly, Ni++ ions are essential for Urease activity, the key enzyme for H.pylori survival in the acidic environment of the stomach, as well as for a respiratory hydrogenase. Urease makes up to 10% of the total cell protein synthesis, so that nickel homeostasis becomes an essential step for the pathogen survival.

2.1 - HpNikR: a pleiotropic regulator

Helicobacter controls this homeostasis through a nickel regulator, HpNikR, a homologue of NikRs from different bacterial species (including E.coli). However, in H.pylori NikR acts as a pleiotropic regulator, being a rare case of master switching regulator in bacteria. Its strong reliance on nickel concentrations and its multiple responsive elements, both positively or negatively controlled, make us think of developing a synthetic regulon for nickel sensing.

nikrFor: gtttcttcgaattcgcggccgcttctagATGGATACACCCAATAAAGACG
nikrRev: gtttcttcctgcagcggccgctactagtattattaCTATTCATTGTGTTCAAAG

(Here the PCR electrophoretic profile). Thus we obtained BBa_K1151000, and its derivative coding device, pLac-regulated BBa_K1151006.

2.2 - HpNikR-controlled promoters

HpNikR, as said above, has multiple target promoters, some of them being upregulated, some being downregulated, in response to nickel concentration in the environment.

2.2.1 - Negative promoters

As shown above, nickel-bound NikR can downregulate the expression of various targets. We obtained a genomic region from H.pylori G27 containing a divergent operon with two NikR-regulated promoters. The plasmid was sent us again by prof. Alberto Danielli as pNKTB. The promoters control the expression of the exbBD operon (for siderophore uptake, called Pexb) and for nikR expression itself (PnikR). According to the functional segmentation of the divergent operon (as in Delany et al., see bibliography below), we tried to separate the promoters, using the following primers (including Biobrick Prefix and Suffix, lowercase):

IntergenicaFor: gtttcttcgaattcgcggccgcttctagagTGAGAAAAATCCTTTTTTG
pnikrev: gtttcttcctgcagcggccgctactagtaTGAGAAAAATCCTTTTTTG
pnikfor: gtttcttcgaattcgcggccgcttctagagAATTCAAACGCTCTTATG
pexbfor: gtttcttcgaattcgcggccgcttctagagACTGGATTTAAATGGTTG
pexbrev:gtttcttcctgcagcggccgctactagtaGCACCCTATAAGAAGGCATC

(Here the PCR Results) So we obtained the whole divergent operon and Pexb and Pnikr. These promoters were cloned upstream of BBa_E0240, becoming parts BBa_K1151009,BBa_K1151010, BBa_K1151011.
Here you can see (link to experimental data) characterisation data for BBa_K1151036 and BBa_K1151038: constructs made up by the promoters cloned upstream of GFP cds together with BBa_K1151006, HpNikR coding device.

2.2.2 - Positive promoter

We also tried a positively-regulated promoter, from UreABCDE operon (PureA). We got it synthetised from IDT, obtaining BBa_K1151005.
We weren't able to characterise BBa_K1151005 activity.

3 - Storage system

The second part of our project aimed to develop a bacterial population able to store nickel, basing its activity on the Hpn protein from H. pylori and a Quorum Sensing signalling: this last natural process is the basis of interaction between the two bacterial communities. The storage system of our design allows to accumulate the metal by bioconcentration: once accumulation occurred, it is necessary to dispose of the bacterial biomass containing the metal. See the Applications page for a design of purification plant implementation.

3.1 - Hpn protein

Hpn is an histidine-rich protein whose role is to store Nickel ions in the cytoplasm of Helicobacter. In conditions of Nickel starvation, this protein supplies the ions to the nickel-dependent machineries. We thought we can use this protein to make E.coli accumulate nickel. Hpn has been demonstrated not to be toxic even if highly induced in this heterologous system (Ge et al., see Bibliography). Hpn coding sequence has been synthetised by IDT, and submitted as BBa_K1151001, and its derivative translational unit BBa_K1151004, generator BBa_K1151025 and coding device BBa_K1151013. We weren't able to characterise these parts activities.

4 - Nickel-dependent Quorum Sensing Network

At first, our project was designed to work on a single plasmid, for both the sensing and metal-accumulation functions. To increase the safety of our device, giving more control to the human operator, we chose to divide the two phases of the project into two different bacterial populations. The activation of the storage depends on the activation of the sensing population. So, in order to integrate the two systems, we exploited the mechanism of intercellular communication of Quorum Sensing. Evaluating the effectiveness of operation of various QS BioBricks present in the Registry, we chose BBa_K805016, BBa_C0261 and BBa_R0062, from Alivibrio fischerii, based and dependent on the synthesis of AHLs as signaling molecules.
In our system, the communication start is Ni2+ and IPTG dependent, both necessary for the synthesis of HpNikR, the genic master regulator which activates the translation of LuxI, the QS inducer. The LuxI protein catalyzes the synthesis of [N-]acyl-homoserine lactones (AHLs), the signalling molecule, from S-adenosylmethionine (SAM) in the sensing population. Quorum sensing molecules reach the other bacterial population by diffusion.
Here we built a system designed on the principle of positive feedback. In fact, the storage population integrates the gene LuxR within the plasmid we designed: LuxR codes for the receiver protein. Placing the transcription of the LuxI gene under the control of the promoter LuxPR, we obtained an AHL signal amplification phenomenon.

Under the same promoter and within the same synthetic operon we also placed the gene of the nickel-binding protein, Hpn, thereby obtaining a triple control of the protein synthesis process in the accumulator population, that depends upon the concentrations of Ni2+, IPTG and AHLs.

5 - General working scheme

Click on the image to zoom!

6 - Bibliography

• Abraham et al, The metal- and DNA-binding activities of Helicobacter pylori NikR. J Inorg Biochem. 2006; 100(5-6): 1005-14.
• Benanti et al, Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters. J Biol Chem. 2011; 18;286: 15728–15737.
• Contreras et al, Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori; Molecular Microbiology, 49 (4) (2003), 947–963.
• Delany et al, In vitro analysis of protein-operator interactions of the NikR and Fur Metal-Responsive Regulators of coregulated genes in Helicobacter pylori. J Bacteriol. 2005 Nov;187(22):7703-15.
• Danielli et al, Built shallow to maintain homeostasis and persistent infection: insight into the transcriptional regulatory network of the gastric human pathogen Helicobacter pylori. PLoS Pathog. 2010, 10; 6(6):e1000938.
• Danielli and Scarlato, Regulatory circuits in Helicobacter pylori: network motifs and regulators involved in metal-dependent responses. FEMS Microbiol Rev, 2010; 34(5):738-52.
• Denkhaus and Salnikow, Nickel essentiality, toxicity and carcinogenity; Critical Reviews in Oncology/Hematology, 42 (2002) 35–56.
• Dian et al, Structural Basis of the Nickel Response in Helicobacter pylori: Crystal Structures of HpNikR in Apo and Nickel-bound States. J Mol Biol. 2006; 25;361(4):715-30.
• Dosanjh et al, Helicobacter pylori NikR’s Interaction with DNA: A Two-Tiered Mode of Recognition. Biochem. 2009, 48, 527–536.
• Dosanjh and  Michel, Microbial nickel metalloregulation: NikRs for nickel ions. Curr Opin Chem Biol. 2006 (2):123-30.
• Ernst et al, The nickel-responsive regulator NikR controls activation and repression of gene transcription in Helicobacter pylori. Infect Immun. 2005; 73(11):7252-8.
• Gadd, Metals, minerals and microbes: geomicrobiology and bioremediation; Microbiology, 156 (2010), 609–643.
• Ge et al, Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori; Biochemistry Journal 393 (2006), 285–293.
• Ge et al, Thermodynamic and kinetic aspects of metal binding to the histidine-rich protein, Hpn. J Am Chem Soc. 2006; 128(35): 11330-1.
• Gilbert et al, Protein Hpn: cloning and characterization of a Histidine-Rich Metal-Binding Polypeptide in Helicobacter pylori and Helicobacter mustelae. Infect Immun. 1995 Jul; 63(7):2682-8.
• Muller et al, Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori. Nucleic Acids Res. 2011; 39(17):7564-75.
• Mutsunori et al, Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli–H. pylori rpoD gene in E. coli. Gene 239 (1999) 351–359.
• “Nickel, Elemental” on TOXNET- Databases on toxicology, hazardous chemicals, environmental health, and toxic releases.
• Seshadri et al, Roles of His-Rich Hpn and Hpn-Like Proteins in Helicobacter pylori Nickel Physiology. J Bacteriol. 2007 Jun; 189 (11): 4120-6.
• van Vliet et al, Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level. Infect Immun. 2001 Aug;69(8):4891-7.
• Zambelli et al, High affinity Ni2+ binding selectively promotes binding of Helicobacter pylori NikR to its target urease promoter. J Mol Biol. 2008 Nov 28;383(5):1129-43.