University of Dundee

Professor Frank Sargent FRSE

Assembly and targeting of complex bacterial enzymes
Position: 
Professor of Bacterial Physiology
Address: 
School of Life Sciences, University of Dundee, Dundee
Full Telephone: 
+44 (0) 1382 386463, int ext 86463
Email: 

Research

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.

This work is funded by the BBSRC and our main collaborators are Prof Fraser Armstrong (University of Oxford), Dr Alison Parkin (University of York), Prof Gary Sawers (Martin-Luther-Universität, Halle-Wittenberg) and Profs Bill Hunter and Tracy Palmer (University of Dundee).

 

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.

Our work in this area is funded by the MRC and our main collaborators are Prof Bill Hunter, Prof Tracy Palmer and Dr David Norman (all University of Dundee) and Prof Geerten Vuister (University of Leicester).

 

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.

This work is currently being undertaken by MRC- and CAPES-funded PhD students and our main collaborators are Dr Sarah Coulthurst, Dr Nicola Stanley-Wall and Prof Tracy Palmer (all University of Dundee).

Teaching

Associate Dean, Research-Led Teaching

 

Organiser, Dundee iGEM Teams

 

 

 

 

Honours Tutor (Molecular Biology Degree Board)

Honours Project - LAB050 Bacterial Membrane Biology

BI40051 - 4B01 Bacterial Membrane Biology

BI40052 - 4C18 Bioenergy & Bioremediation

BS31004 - Biochemistry & Cell Biology

BS31005 - Genetics

BS32004 - Molecular Microbiology

BS22003 - Laboratory & Research Skills C

 

External examiner, University of York

Publications

 
Bowman, L., Balbach, J., Walton, J., Sargent, F. and Parkin, A. (2016) Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 21, 865-873
 
Carr, S. B., Evans, R. M., Brooke, E. J., Wehlin, S. A., Nomerotskaia, E., Sargent, F., Armstrong, F. A. and Phillips, S. E. (2016) Hydrogen activation by [NiFe]-hydrogenases. Biochemical Society transactions. 44, 863-868
 
Connelly, K. R., Stevenson, C., Kneuper, H. and Sargent, F. (2016) Biosynthesis of selenate reductase in Salmonella enterica: critical roles for the signal peptide and DmsD. Microbiology. 162, 2136-2146
 
Evans, R. M., Brooke, E. J., Wehlin, S. A., Nomerotskaia, E., Sargent, F., Carr, S. B., Phillips, S. E. and Armstrong, F. A. (2016) Mechanism of hydrogen activation by [NiFe] hydrogenases. Nature chemical biology. 12, 46-50
 
Foulkes, J. M., Deplanche, K., Sargent, F., Macaskie, L. E. and Lloyd, J. R. (2016) A Novel Aerobic Mechanism for Reductive Palladium Biomineralization and Recovery by Escherichia coli. Geomicrobiology Journal. 33, 230-236
 
Pinske, C. and Sargent, F. (2016) Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid. MicrobiologyOpend.o.i: 
 
Sargent, F. (2016) The Model [NiFe]-Hydrogenases of Escherichia coli. Advances in microbial physiology. 68, 433-507
 
Wulff, P., Thomas, C., Sargent, F. and Armstrong, F. A. (2016) How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 21, 121-134
 
Kelly, C. L., Pinske, C., Murphy, B. J., Parkin, A., Armstrong, F., Palmer, T. and Sargent, F. (2015) Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of. Biotechnology reports. 8, 94-104
 
McDowall, J. S., Hjersing, M. C., Palmer, T. and Sargent, F. (2015) Dissection and engineering of the Escherichia coli formate hydrogenlyase complex. FEBS Lett. 589, 3141-3147
 
Pinske, C., Jaroschinsky, M., Linek, S., Kelly, C. L., Sargent, F. and Sawers, R. G. (2015) Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli. J Bacteriol. 197, 296-306
 
Pinske, C., Sargent, F. and Sawers, R. G. (2015) SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells. Metallomics. 7, 683-690
 
Bowman, L., Flanagan, L., Fyfe, P. K., Parkin, A., Hunter, W. N. and Sargent, F. (2014) How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase. Biochem J. 458, 449-458
 
Dow, J. M., Grahl, S., Ward, R., Evans, R., Byron, O., Norman, D. G., Palmer, T. and Sargent, F. (2014) Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone. The FEBS journal. 281, 246-260
 
Hamilton, J. J., Marlow, V. L., Owen, R. A., Costa Mde, A., Guo, M., Buchanan, G., Chandra, G., Trost, M., Coulthurst, S. J., Palmer, T., Stanley-Wall, N. R. and Sargent, F. (2014) A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens. J Cell Biol. 207, 615-626
 
McDowall, J. S., Murphy, B. J., Haumann, M., Palmer, T., Armstrong, F. A. and Sargent, F. (2014) Bacterial formate hydrogenlyase complex. Proc Natl Acad Sci U S A. 111, E3948-3956
 
Murphy, B. J., Sargent, F. and Armstrong, F. A. (2014) Transforming an oxygen-tolerant [NiFe] uptake hydrogenase into a proficient, reversible hydrogen producer. Energ Environ Sci. 7, 1426-1433
 
Wulff, P., Day, C. C., Sargent, F. and Armstrong, F. A. (2014) How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases. P Natl Acad Sci USA. 111, 6606-6611
 
Bowman, L., Palmer, T. and Sargent, F. (2013) A regulatory domain controls the transport activity of a twin-arginine signal peptide. FEBS Lett. 587, 3365-3370
 
Dow, J. M., Gabel, F., Sargent, F. and Palmer, T. (2013) Characterization of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway. Biochem J. 452, 57-66
 
Evans, R. M., Parkin, A., Roessler, M. M., Murphy, B. J., Adamson, H., Lukey, M. J., Sargent, F., Volbeda, A., Fontecilla-Camps, J. C. and Armstrong, F. A. (2013) Principles of sustained enzymatic hydrogen oxidation in the presence of oxygen--the crucial influence of high potential Fe-S clusters in the electron relay of [NiFe]-hydrogenases. Journal of the American Chemical Society. 135, 2694-2707
 
James, M. J., Coulthurst, S. J., Palmer, T. and Sargent, F. (2013) Signal peptide etiquette during assembly of a complex respiratory enzyme. Mol Microbiol. 90, 400-414
 
Sargent, F., Davidson, F. A., Kelly, C. L., Binny, R., Christodoulides, N., Gibson, D., Johansson, E., Kozyrska, K., Lado, L. L., Maccallum, J., Montague, R., Ortmann, B., Owen, R., Coulthurst, S. J., Dupuy, L., Prescott, A. R. and Palmer, T. (2013) A synthetic system for expression of components of a bacterial microcompartment. Microbiology. 159, 2427-2436
 
Volbeda, A., Darnault, C., Parkin, A., Sargent, F., Armstrong, F. A. and Fontecilla-Camps, J. C. (2013) Crystal structure of the O(2)-tolerant membrane-bound hydrogenase 1 from Escherichia coli in complex with its cognate cytochrome b. Structure. 21, 184-190
 
Coulthurst, S. J., Dawson, A., Hunter, W. N. and Sargent, F. (2012) Conserved signal peptide recognition systems across the prokaryotic domains. Biochemistry. 51, 1678-1686
 
Grahl, S., Maillard, J., Spronk, C. A., Vuister, G. W. and Sargent, F. (2012) Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide. Mol Microbiol. 83, 1254-1267
 
Parkin, A., Bowman, L., Roessler, M. M., Davies, R. A., Palmer, T., Armstrong, F. A. and Sargent, F. (2012) How Salmonella oxidises H(2) under aerobic conditions. FEBS Lett. 586, 536-544
 
Parkin, A. and Sargent, F. (2012) The hows and whys of aerobic H2 metabolism. Current opinion in chemical biology. 16, 26-34
 
Pinske, C., Jaroschinsky, M., Sargent, F. and Sawers, G. (2012) Zymographic differentiation of [NiFe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12. BMC Microbiol. 12, 134
 
Pinske, C., McDowall, J. S., Sargent, F. and Sawers, R. G. (2012) Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12. Microbiology. 158, 856-868
 
 
 

Impact

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.