Professor Frank Sargent FRSE
1. Bioenergy & Industrial Biotechnology – understanding and engineering hydrogenases and hydrogen metabolism.
Work in this area concentrates on the molecular biology of bacterial hydrogen metabolism. Escherichia coli and Salmonella enterica are related bacteria that can use hydrogen as a respiratory electron donor, and can generate hydrogen gas under fermentative conditions. Central to these activities are the nickel-dependent hydrogenases. We are currently studying the structure and function of a special group of oxygen-tolerant [NiFe] hydrogenases. This family enzymes, which include the E. coli Hyd-1 and Salmonella Hyd-5 isoenzymes, contain a unique [4Fe-3S] metal cofactor, the chemistry of which helps to protect the active site from oxygen attack. In addition, we are studying the structure and function of the bacterial formate hydrogenlyase (FHL) complex, which is responsible for the majority of hydrogen production from enteric bacteria.
A separate project aims to take a synthetic biology approach to enhancing hydrogen production by microbes. Hydrogen has the highest energy per weight of any fuel and is versatile, since it can also be used in fuel cells. Biohydrogen offers the prospect of fully renewable hydrogen, freed from any dependence on fossil fuel. The problems are that biohydrogen production by bacteria is usually of low yields and at low rates. Protein engineering, metabolic engineering, and directed evolution techniques are being integrated to design and build synthetic enzymes and strains that will address these issues.
2. The bacterial Tat pathway – the structure and function of signal peptides and their binding proteins.
Respiratory enzymes are complex enzymes comprising many protein subunits containing any number of prosthetic groups. Moreover, these enzymes are often buried in the cell membranes or located completely outside the cytoplasm. As a result, biosynthesis of complex respiratory enzymes requires the coordination of cofactor insertion, protein folding, and protein targeting processes. A subset of respiratory enzymes are synthesised as precursors with N-terminal signal peptides bearing a conserved SRRxFLK ‘twin-arginine’ amino acid motif. Proteins with twin-arginine signal peptides are transported fully folded by an export apparatus termed the twin-arginine translocation (Tat) system. Research in the group currently focuses on novel molecular, biochemical, and applied aspects of Tat-targeted bacterial respiratory enzymes.
We identified a chaperone-mediated ‘Tat proofreading’ process involved in the coordination of protein assembly and export. The paradigm Tat proofreading chaperone is TorD, a small multi-functional protein, which binds tightly to the Tat signal peptide of the molybdenum-dependent trimethylamine N-oxide reductase thus preventing premature export of the enzyme until all assembly proceses are complete. TorD-like chaperones are almost ubiquitous in bacteria and archaea, and can be found directing assembly of selenate reductases (e.g. DmsD) and tetrathionate reductases (e.g. TtrD). However, TorD family proteins are not the only Tat proofreading chaperones since we have also studied the NapD family, which is a chaperone that binds tightly to the Tat signal peptide of nitrate reductases.
3. Protein secretion in opportunistic pathogens.
One key process by which bacteria adapt to their environment or host is via the secretion of effector molecules, which are deliberately translocated from inside the cell across the cell envelope. Translocation of proteins requires active secretion, for which six different systems have so far been identified in Gram-negative bacteria. Not all bacteria contain all six secretion systems, however it is known that protein secretion can occur in a ‘single step’ (such as in Type I, Type III or Type VI systems) or in a ‘two step’ system (for example Type II or Type V), when the secreted substrate is first targeted to the periplasm, either by the Sec or Tat export machineries, and then further externalised by a secondary outer membrane translocation event.
The Serratia marcescens genome encodes three extracellular chitinases (ChiA, ChiB, and ChiC) and one extracellular chitin binding protein (Cbp21) that have roles in both basic physiology and pathogenicity. Two of these (ChiB and ChiC) contain no obvious targeting signals whatsoever. However, both ChiA and Cbp21 are synthesised with N-terminal Sec signal peptides. It was a possibility that these proteins could be secreted by a two-step Type II system. However, Serratia strains do not always contain typical outer membrane components of Type II systems. The aim of this project is to take genetic, biochemical and whole systems approaches to characterise the complete chitinase secretion pathway.
BS41004 - Level 4 Biological Sciences Project
BS42009 - Level 4 Advanced Molecular Microbiology
BS32012 - Level 3 Synthetic Biology Open Lab
BS32004 - Level 3 Molecular Microbiology
BS31004 - Level 3 Biochemistry and Cell Biology
BS31005 - Level 3 Genetics
Owen,R.A., Fyfe,P.K., Lodge,A., Biboy,J., Vollmer,W., Hunter,W.N., Sargent,F. (2018) The structure and activity of ChiX: a peptidoglycan hydrolayse required for chitinase secretion in Serratia marcescens. Biochemical Journal 475, 415-428.
Roger,M., Brown,F., Gabrielli,W., Sargent,F. (2018) Efficient hydrogen-dependent carbon dioxide reduction by Escherichia coli. Current Biology 28, 140-145.
Lindenstrauß,U., Skorupa,P., McDowall,J.S., Sargent,F., Pinske,C. (2017) The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex. Biochemical Journal 474, 2937-2950.
Lamont,C.M., Kelly,C.L., Pinske,C., Buchanan,G., Palmer, T., Sargent,F. (2017) Expanding the substrates for a bacterial hydrogenlyase reaction. Microbiology 163, 649-653.
Lamont,C.M., Sargent,F. (2017) Design and characterisation of synthetic operons for biohydrogen technology. Archives of Microbiology 199, 495-503.
Albareda,M., Buchanan,G., Sargent,F. (2017) Identification of a stable complex between a [NiFe]-hydrogenase catalytic subunit and its maturation protease. FEBS Letters 591, 338-347.
Connelly,K.R.S., Stevenson,C., Kneuper,H., Sargent,F. (2016) Biosynthesis of selenate reductase in Salmonella enterica: critical roles for the signal peptide and DmsD. Microbiology 162, 2136-2146.
Pinske,C., Sargent,F. (2016) Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid. Microbiology Open 5, 721-737.
The hydrogen and hydrogenases research that we do is particularly well suited to public engagement activities, and also has the potential to be spun out into biomedical and biotechnological enterprises.
Since 2010 our divisional biennial “Magnificent Microbes” event at Dundee Science Centre has featured an interactive biohydrogen stall aimed at explaining how sugar-rich food- and agricultural-wastes can be used to make biofuels, including hydrogen. This stall, which features a bioreactor producing hydrogen that can then be converted into electricity by a fuel cell, has had frequent outings at the Dundee Doors Open Days, Dundee Family Fun Days, Café Science, as well as both the Dundee and Edinburgh Science Festivals. These events allows us to explain the importance of research into alternative energy sources, and exposes the public to modern new ways of working, such as aspects of synthetic biology, computational biology, and mathematical modelling.