The essential role of copper in bacterial methane oxidation: characterising a novel copper protein involved in storage and the soluble copper proteome

Methane-oxidising bacteria use methane, a potent greenhouse gas, as a source of carbon and energy. To enable these organisms to perform this key function most effectively they require large quantities of copper. Methane-oxidising bacteria have evolved novel mechanisms that ensure they are able to accumulate the copper they need to metabolise methane. This includes the secretion of small peptides called methanobactins (mbtins) that are able to sequester the minute amounts of copper present in the organism's surrounding environment. Methane-oxidising bacteria use mbtin to uptake copper, with one of the main uses being for the enzyme that oxidises methane (a methane monooxygenase). How these organisms handle copper, which is potentially toxic as well as being essential, has not been studied in any detail. Whilst analysing extracts from Methylosinus trichosporium OB3b, a methane-oxidising bacterium, for mbtin we have found a number of high abundance soluble copper pools containing proteins that bind this metal. The presence of large amounts of soluble copper in M. trichosporium OB3b was unanticipated as only two low-abundance soluble copper proteins are predicted (the copper-containing methane monooxygenase is membrane bound). One of the high abundance copper pools has been found to contain a novel copper protein, potentially involved in storing copper ions, herein called Csp1. The other soluble copper pools have not yet been studied in any detail.

In the proposed work we will study copper binding to Csp1 both in M. trichosporium OB3b and also using protein that we have produced in large amounts via an over-expression host. We will analyse the structure of Csp1 and how this is influenced by copper binding. We will investigate the role of Csp1 in copper storage by genetically engineering M. trichosporium OB3b, as well as by looking at how copper levels during growth influence the production of Csp1. Proteins similar to Csp1 are present in methane-oxidising bacteria and also in other bacteria. The Csp1 homologues from M. trichosporium OB3b and Bacillus subtilis, an organism in which copper handling has been studied and that is more tractable for genetic engineering approaches, will be studied. The other soluble copper pools that have been identified in M. trichosporium OB3b will also be analysed with the aim of finding additional novel copper-binding proteins. The proposed studies will provide insight into copper management in a relatively simple system of environmental importance and therefore has applications in helping to understand the oxidation of methane in Nature and the mitigation of the contribution this greenhouse gas makes to global warming. Furthermore, vast gaseous methane reserves are untapped as a feedstock for liquid fuels and chemical production due to the expense and difficulties associated with the available processes that can facilitate the conversion to methanol, a reaction readily performed by methane-oxidising bacteria. Understanding how these organisms handle an essential cofactor, copper, for the oxidation of methane therefore has potential biotechnological applications.

Methane-oxidising bacteria use copper to convert methane to methanol as the first step of growth on this carbon and energy source. Copper is the essential cofactor of the most efficient particulate methane monooxygenase. Copper is also potentially toxic and methane-oxidising bacteria have evolved novel mechanisms to handle this metal. Methane-oxidising bacteria secrete small copper-binding peptides called methanobactins (mbtins) under low copper conditions that deliver the metal to the cytoplasm. Most bacteria do not require copper in this compartment. Whilst looking for internalised mbtin in the methane-oxidising bacterium Methylosinus trichosporium OB3b we have found a number of high abundance soluble copper pools. One of these contains a novel copper protein potentially involved in storing copper, herein called Csp1, which has a predicted Tat signal sequence and is therefore expected to be exported from the cytoplasm folded.

We will investigate the ability of Csp1 to bind Cu(I) both in M. trichosporium OB3b and also in vitro using recombinant protein. Csp1 is predicted to be a 4-helix bundle with all 13 Cys residues pointing towards the core of the protein. The number of Cu(I) equivalents, and other metals such as Zn(II), that can be accommodated within this bundle will be determined as will metal affinities. The structures of apo and metal-loaded protein will be analysed. The in vivo function of Csp1 will be investigated using a Csp1-delete strain and also by analysing if the csp1 gene is transcriptionally regulated by copper.

Non-Tat homologues of Csp1, that are expected to be cytosolic, are present in methane-oxidising and other bacteria. We will investigate this protein in M. trichosporium OB3b and also Bacillus subtilis. We will analyse the other soluble copper pools that we have found in M. trichosporium OB3b with the aim of identifying further novel copper proteins. Time permitting, these proteins will also be investigated.

The link between copper and the oxidation of methane by bacteria makes this work highly conducive to outreach activities. The proposed studies also have numerous potential applications. Understanding how copper handling influences methane oxidation by these organisms will help in the development of biological methods for mitigating the emission of this important greenhouse gas into the atmosphere (strategic priority; living with environmental change). Vast reserves of methane gas are currently underutilised for the production of liquid fuels and chemicals due to the cost and difficulty associated with a reaction that methane-oxidising bacteria readily perform with the help of copper (highlight area: industrial biotechnology and bioenergy, strategic priority: bioenergy; generating new and replacement fuels for a greener, sustainable future). Furthermore, the broad specificity of methane monooxygenase makes these organisms useful for bioremediation applications (highlight area: industrial biotechnology and bioenergy).

Impact is assisted by the fact that copper-containing compounds are widely used for their ability to prevent the growth of a range of organisms in various applications, including the addition of copper to anti-fouling paints, the use of copper, along with silver to disinfect water, the use of copper in hospital fixtures to reduce transmission of nosocomial infections, and copper-based agrochemicals. Additionally, copper mis-handling is associated with a range of diseases. Many biotechnologically useful proteins (highlight area: industrial biotechnology and bioenergy) and biopharmaceuticals are metalloproteins and their recombinant production requires correct metallation. Furthermore, in synthetic biology approaches, another BBSRC strategic priority, the production of modified organisms with a gain of function acquired through the addition of a particular metalloprotein, for example a methane monooxygenase, would require this protein to be correctly metallated.

The Investigators have been involved in a range of outreach activities including local schools and the wider public. The results of research are shared via personal web pages and Institute and University web sites, and press releases when appropriate. Findings can be publicised through various routes including general scientific meetings/exhibitions attended by public and industrial audiences and the wider scientific community. Assistance is available to further develop outreach activities via Beacon NE, which is a partnership between Newcastle University, Durham University and the Centre for Life (Newcastle) that provides training and development opportunities in public engagement.

Numerous contacts already exist with the Industrial sector. Results will be regularly assessed to identify any IP and/or commercially exploitable outcomes via meetings with the Newcastle University Commercial Development Team. If the results we obtain about copper handling by methane-oxidising bacteria are of benefit to any of the applications mentioned above we will publicise this to the relevant businesses. We will transmit technology, in terms of knowledge and techniques, through publication in high impact factor journals and by presentations at international conferences.

The RA working on this project will benefit from improved skills, knowledge and experience gained from the research, and also from the associated training. The RA will be encouraged to contribute to outreach activities. This work will also benefit other members of the Investigators' groups, and particularly PhD students, through exposure to new techniques and developing theories. Academic audiences will be reached via publications and presentations at international conferences and collaborations.