Team:UniSalento Lecce

Introduction to NickBusters Project

Nickel is one of the most widespread heavy metals in the ecosystem, and it is essential for many organisms, from bacteria to higher forms of life. However, as all the heavy metals, even a small amount of Nickel higher than the essential dose could be toxic, leading to various noxious effects [1-2]: then the need to remove its excess from many substrates. Nowadays bacteria-mediated bioremediation from inorganic substances seems to be a considerably relevant frontier in microbic biotechnologies [3]. Our project aims to develop a living system, in an easy monitorable bacterial platform, who would work as a Nickel detector and a Nickel remediation system. The whole system is based on genetic parts from the pathogen Helicobacter pylori, whose evolution led to a stronger reliance on Nickel ions for host interaction and pathogenesis. Sensing relies on nickel regulator NikR from H. pylori (HpNikR): though homolog of E.coli NikR, it has a stronger dependance on Nickel ions and a wider range of regulated elements, being a rare case of pleiotropic regulator in bacteria. As HpNikR could contemporaneously activate and repress gene expression [4-5-6], we thought of developing a synthetic gene cluster under HpNikR - regulated promoters. With a double regulation, one of the promoters being activated while the other being repressed, the system allows to follow up Nickel sensing as a fluorescence signal from fluorescent proteins. Thus, adding bacteria to a nickel containing medium results in switching off a basal occuring green fluorescence and in switching on a red fluorescence signal.

The Nickel storage system is even taken from our pathogenic friend: Hpn protein is a Nickel binding protein, whose role is to store the Nickel ions inside the cell and to supply them to other proteic interactors, from Nickel dependent heat shock proteins to pathogenesis factors as urease [7]. Hpn can be expressed in a heterologous model system (E.coli), increasing bacterial resistance to toxic dose of Nickel ions and allowing Ni++ intracellular accumulation [8]. We thought to split the sense and store machineries in two different bacterial populations, making them communicate through Quorum Sensing [9]: we used the LuxI/LuxR system from Alivibrio fischerii. The first population senses nickel concentration in medium ( and translates it in a fluorescence signal), then activating Acyl-Homoserine-Lactones (AHLs) production. The second population receives the QS signal and sums it up to the nickel detection signal (as in an AND gate), activating Hpn (the storage protein) expression and AHLs signal amplification.

Bibliography

1. Denkhaus and Salnikow, Nickel essentiality, toxicity and carcinogenity; Critical Reviews in Oncology/Hematology, 42 (2002) 35–56
2. “Nickel, Elemental” on TOXNET- Databases on toxicology, hazardous chemicals, environmental health, and toxic releases.
3. Gadd, Metals, minerals and microbes: geomicrobiology and bioremediation; Microbiology, 156 (2010), 609–643
4. Dosanjh and Michel, Microbial nickel metalloregulation: NikRs for nickel ions; Current Opinion in Chemical Biology, 10 (2006), 123–130
5. Contreras et al., Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori; Molecular Microbiology, 49 (4) (2003), 947–963
6. Muller et al., Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori; Nucleic Acids Research, 39 (17) (2011), 7564–7575
7. Seshadri et al., Roles of His-Rich Hpn and Hpn-Like Proteins in Helicobacter pylori Nickel Physiology; Journal of Bacteriology, 189 (11) (2007), 4120–4126
8. 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
9. Miller, Bassler, Quorum Sensing in bacteria; Annual Review of Microbiology 55 (2001), 165-99.