The discernment of metals by a set of DNA-binding transcriptional regulators

It has recently been estimated that 47% of all enzymes require metals such as copper, zinc, nickel, cobalt, iron, manganese, calcium and magnesium. On average, almost half of all attempts to manipulate the activities of cells (for example in metabolic engineering) will involve an enzyme which must somehow acquire the correct metal. Selection of the correct metals by enzymes is substantially governed by metal-availability at the site of protein folding, and metal-availability in cells is, in turn, substantially governed by sensors that detect excess or deficiency of each metal. Crucially, these sensors somehow discern the different inorganic elements, one from another. A zinc-sensor called MTF1 is currently the only DNA-binding, metal-binding, metal-sensor known in humans. However, over the last two decades we, and many others, have discovered an expanding repertoire of bacterial DNA-binding metal-sensing proteins. These sensors turn genes on or off; each sensor acting in response to specific metals. The genes that some of the sensors regulate have been found and the metals they respond to identified. Among the regulated genes are ones encoding importers that acquire more of those metals which are needed and exporters that pump out metals that are surplus to requirements and/or solely toxic. The sensors work in several different ways. Some bind to DNA and, in effect, switch a gene off. When the sensor binds to the metal its structure changes such that it no longer binds tightly to the DNA and the gene becomes active. Other metal-sensors do the reverse. Their structure changes upon binding a metal such that only under these conditions do they bind tightly to DNA and switch a gene off. Finally, some sensors bind to DNA both with and without a metal but a change in protein structure caused by binding the metal distorts the DNA to activate gene expression. The characterisation of these assorted metal sensors has provided an opportunity to explore how metals are discerned. A naïve expectation was that each sensor would tightly bind the metal it detected and bind all other metals weakly or not at all. But this turns out not to be the case and indeed fundamental rules of bioinorganic chemistry imply that for flexible proteins it could rarely be the case. Thus the question becomes, regardless of the mechanism of gene regulation, how does each metal trigger the correct sensor protein? To answer this question we need to consider a set of sensors from a single bacterium. We need to consider their affinities for different metals, not in isolation, but in the context of the metal-affinities of all of the other metal-sensors in the same cell. A simple explanation could be that the sensors give the correct integrated response as a function of their 'relative' metal affinities rather than their 'absolute' metal affinities: the cobalt sensor being the tightest cobalt-binder of the set, the zinc-sensor being the tightest zinc-binder of the set, and so on. The organism chosen for this work, a cyanobacterium, has a set of metal-sensors with properties that are peculiarly well suited to comparing their metal-affinities, one against the other, using a method that we exploited and published in 2007. In this program we will also characterize at least one new metal-sensor. This is fundamental research. Discerning metals is, literally, elemental to life. Nonetheless, it has implications and applications across the biosciences and biotechnology and for this reason we, and the rest of the 'Metals in Cells' group at Newcastle, actively collaborate with the biotechnology industrial sector. Industrial links related to this programme are described in the impact plan.

The aim is to discover how a cell discerns metals. At least seven families of DNA-binding, cytosolic metal sensors have been characterised in bacteria (Fur, DtxR, MerR, CsoR-RcnR, ArsR-SmtB, CopY, NikR). The exact complement of metal-sensors varies between bacteria with different representatives of each sensor-family responding to different metals. We need to understand how the correct sensor among the complement of metal sensors in a given cytosol responds to the correct metal. A novel hypothetical explanation for metal-specificity will be tested. The cyanobacterium Synechocystis PCC 6803 has been selected for this work due to (i) properties of its set of known, or deduced, metal-sensors which are peculiarly favourable for some of the planned experiments (ii) our background knowledge, biological reagents and technical expertise, (iii) the special physiological significance and biotechnological implications of metals such as copper, iron, nickel and cobalt in these organisms. This is an opportunity to obtain a comprehensible answer to the question of how a network of receptors is integrated to give rise to the correct cellular responses. The actions of at least one new metal-sensor will also be characterised in the course of this research.

By controlling the levels of metals in cells metal sensors affect metal occupancy of metallo-proteins. Since some 47% of enzymes require metals, understanding how sensors discern metals, one from another, has broad implications across the biosciences and broad applications in biotechnology. Considerable time and energy has been expended in the past ~ six months to establish valuable industrial links and some of these are described in the (confidential) impact summary. Letters of support are attached. The most imminent of the anticipated applications relates to bio-processing and the production of recombinant metallo-proteins. Here the timescale for possible marketed products could be five years. Many synthetic biology projects are liable to require the manipulation of metal supply. Of these, engineering the supply of nickel for hydrogenase for biohydrogen production by photosynthetic bacteria is perhaps the most urgent. We know that nickel supply is poor in cyanobacteria and that this limits hydrogenase activity. The PI is a member of the Sir Joseph Swan Institute for Energy Research and collaborations have been established with individuals and networks involved in developing the photoproduction of hydrogen. Here the timescales for benefits to be fully realised, in terms of hydrogen fuel, are presumed to exceed ten years. The detection of metals by pathogens is central to virulence. Some metals and metal-chelators are already in use both as agrochemicals for crop and livestock protection, and in therapies related to human health. It is expected that the knowledge gained from this project could be applied to the development of new agrochemicals and new therapeutics. The development cycle for new agrochemicals and therapeutics is long and time to market is unlikely to be less than ten years. Nonetheless, there is scope for optimising use of existing treatments, perhaps in novel combinations, and here the lead time may be much swifter.