Portrait of Dr Mark Shepherd

Dr Mark Shepherd

Senior Lecturer in Microbial Biochemistry
Programme Director: MSc Infectious Diseases


Dr. Mark Shepherd joined the School of Biosciences in 2011. He studied Biochemistry at the University of Sheffield and stayed in the Department of Molecular Biology & Biotechnology to undertake a PhD with Professor Neil Hunter FRS (1999-2003), where he developed his interest in the enzymology and spectroscopy of chlorophyll and haem biosynthesis. After completing his PhD studies, he conducted postdoctoral research with Prof. Harry Dailey at the University of Georgia, where his research focussed on the terminal enzymes of haem biosynthesis. This was followed by a move back to the University of Sheffield in 2005 to take up a postdoctoral position with Prof. Robert Poole, where he developed interests in E. coli respiration, globin proteins, and the response of bacteria to nitric oxide. A subsequent position at the University of Queensland with Prof. Mark Schembri focussed on nitric oxide stress in Uropathogenic E. coli. In 2011 he moved to the University of Kent, where he is now a Senior Lecturer in Microbial Biochemistry.

Research interests

Topics of interest include antibiotic resistance, stress tolerance, respiratory metabolism, haem biosynthesis, disulphide folding, and biofuel production. A collaborative approach is adopted using a wide variety of techniques including chromosomal engineering, transcriptomics, proteomics, metabolomics, microscopy, protein biochemistry, enzyme kinetics, and structural biology.
Current Projects:
Antimicrobial resistance in E. coli clinical isolates
E. coli causes serious conditions including sepsis, bladder infections, kidney failure, and dysentery. The emergence of drug resistance in pathogenic strains is an increasing problem on a global scale. We liaise with collaborators in the NHS to collect E. coli clinical isolates for antibiotic screening and for other phenotypic characterisation. Collaborative genome sequencing approaches are undertaken to provide insights into the prevalence, evolution, and spread of antibiotic resistance in this deadly human pathogen.
Adaptations to nitric oxide (NO) in uropathogenic E. coli
Uropathogenic E. coli (UPEC) strains are a major cause of urinary tract infections (UTIs), and emergence of antibiotic resistance in UPEC is becoming an increasingly serious problem. Of special interest is the response of E. coli to nitric oxide (NO), a remarkable regulatory molecule and a powerful tool used by mammalian cells to combat microbial infection. Bacteria encounter nitric oxide from host NO synthases in the gut, and NO is also encountered by UPEC in the human urinary tract during UTI. The toxic effects of NO to bacterial cells are diverse: NO can readily diffuse across cell membranes to react with various targets, including haems, iron-sulphur clusters, and protein thiols. Invading E. coli respond in a variety of ways that confer tolerance to NO exposure and enable persistence in the host. The overarching goal is to advance the understanding of NO tolerance mechanisms in drug-resistant UPEC strains.
Bacterial disulphide bond formation
Proteins must be assembled and correctly folded to function, and a key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues. Many bacterial virulence factors, such as fimbriae, flagella, and toxins either contain disulphide bonds or require them in some component of their assembly pathway. In addition, the yield and quality of disulphide-containing protein therapeutics produced in E. coli, such as antibodies and hormones, is influenced by the expression of recombinant folding chaperones. Our current focus is to elucidate the biochemical properties and physiological roles of disulphide chaperones in virulence and survival and to determine their suitability to improve disulphide folding of proteins of biotechnological importance.
Biofuel production in Clostridium
Solventogenic strains of Clostridium are used to produce acetone, butanol and ethanol via the process of ABE fermentation. Besides their use as biofuels, derivatives of ABE fermentation are used to manufacture latex paints, lacquers, enamels, automotive coatings and industrial coatings, and are used are used as solvents in vinyl, cellulosic, acrylics, urethanes and epoxy coatings. We combine strain engineering approaches with metabolomics and genomics to gain a fundamental understanding of Clostridium metabolism, and apply this knowledge to optimise solvent yields.
Bacterial haem synthesis
Bacteria require the cofactor haem for a number of essential cellular processes. Haem biosynthetic enzymes remain a long-term interest in the Shepherd lab, and we have a particular focus on enzymatic metal chelation and porphyrinogen oxidation reactions. We employ a combination of bacterial genetics, bioinformatics, protein biochemistry, enzymology and a variety of spectroscopic approaches to study interests ranging from pathway evolution to mechanistic enzymology. 



  • BI520 – Metabolism & Metabolic Disease 
  • BI521 – Metabolism & Metabolic Regulation (convenor) 
  • BI547 – Plant Physiology & Adaptation
  • BI548 – Microbial Genetics & Physiology I
  • BI600 – Biology Project 
  • BI606 – Pathogens & Pathogenicity BI628 – Microbial Genetics & Physiology II 

Programme Director for MSc Infectious Diseases

  • BI853 – Bacterial Pathogens (convenor)


My group comprises PhD students, MSc students, and technical staff working on a range of projects on bacterial metabolism, biochemistry, genetics and genomics. Applications will be considered from self-funded students (for MSc & PhD) and from postdoctoral researchers intent on securing their own fellowship.

Msc projects available for 2019/20  

Fruit to fuels: assessment of Kent crops as feedstocks for butanol production by Clostridium Joint Supervisor Dr Gary Robinson Strains of Clostridium bacteria can be used to produce butanol, an energy-rich biofuel. This project will assess the ability of Clostridium to convert agricultural/industrial fruit waste from the Kent region into butanol. Techniques: feedstock design, anoxic bacterial culture & fermentation, solvent analysis (GCMS).
Additional research costs: £1200
Engineering microcompartments into biofuel bacteria: new (fluorescent) wheels on an old chassis Joint Supervisor Prof Martin Warren Clostridium bacteria have been used for the industrial production of biofuels for over a century. This project aims to introduce recombinant microcompartments that could be used for improving fuel production or to facilitate the assembly of high-value compounds and proteins. Techniques: cloning, anoxic bacterial growth, fluorescence microscopy, electron microscopy.
Additional research costs: £1500

The CydDC transporter of E. coli: biochemical characterisation of an antimicrobial target Joint supervisor Dr Chris Mulligan CydDC is an ABC transporter that is important for bacterial virulence, resistance to ß-lactam antibiotics, and tolerance of the host immune response. This project aims to gain a better understanding of how CydDC interacts with nitric oxide, a free radical produced by the body in response to infection. Techniques: purification of membrane proteins (nanodiscs), ultracentrifugation, ATPase assays, vesicle reconstitutions, membrane transport assays. 
Additional research costs: £1200


  • Biochemical Society local ambassador
  • Member of the Tetrapyrrole Discussion Group 



  • Skotnicová, P. et al. (2018). The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase. Journal of Biological Chemistry [Online]. Available at: http://dx.doi.org/10.1074/jbc.RA118.003441.
    Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b. The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.
  • Hobbs, C., Reid, J. and Shepherd, M. (2017). The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron binding site. Biochemical Journal [Online] 474:3513-3522. Available at: http://dx.doi.org/10.1042/BCJ20170362.
    The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently-discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.
  • Hobbs, C., Dailey, H. and Shepherd, M. (2016). The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis. Biochemical Journal [Online] 473:3997-4009. Available at: http://dx.doi.org/10.1042/BCJ20160696.
    Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates including non-metallated porphyrins such as protoporphyrin IX, which is well-known to generate reactive oxygen species (ROS) in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Herein, we demonstrate for the first time that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [FeIV=O]+, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction.
  • Holyoake, L. et al. (2016). CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress. Biochemical Journal [Online] 473:693-701. Available at: http://dx.doi.org/10.1042/BJ20150536.
    The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homeostasis, and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), ?-oxidation of fatty acids, and genes involved in nitrate/nitrite reduction. 1H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD+ in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ?pH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism, and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd -type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli , highlight the importance of bacterial redox homeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex.
  • Shepherd, M. et al. (2016). The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection. Scientific reports [Online] 6:35285. Available at: http://dx.doi.org/10.1038/srep35285.
    Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.
  • Holyoake, L., Poole, R. and Shepherd, M. (2015). The CydDC Family of Transporters and Their Roles in Oxidase Assembly and Homeostasis. Advances in Microbial Physiology [Online] 66:1-53. Available at: http://www.dx.doi.org/10.1016/bs.ampbs.2015.04.002.
    The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette type transporter (ABC transporter) that exports the thiol-containing redox-active molecules cysteine and glutathione. These reductants are thought to aid redox homeostasis of the periplasm, permitting correct disulphide folding of periplasmic and secreted proteins. Loss of CydDC results in the periplasm becoming more oxidising and abolishes the assembly of functional bd-type respiratory oxidases that couple the oxidation of ubiquinol to the reduction of oxygen to water. In addition, CydDC-mediated redox control is important for haem ligation during cytochrome c assembly. Given the diverse roles for CydDC in redox homeostasis, respiratory metabolism and the maturation of virulence factors, this ABC transporter is an intriguing system for researchers interested in both the physiology of redox perturbations and the role of low-molecular-weight thiols during infection.
  • Shepherd, M. (2015). The CydDC ABC transporter of Escherichia coli: new roles for a reductant efflux pump. Biochemical Society Transactions [Online] 43:908-912. Available at: http://dx.doi.org/10.1042/BST20150098.
    The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette (ABC) transporter that exports cysteine and glutathione to the periplasm. These reductants are thought to modulate periplasmic redox poise, impacting upon the disulfide folding of periplasmic and secreted proteins involved in bacterial virulence. Diminished CydDC activity abolishes the assembly of functional bd-type respiratory oxidases and perturbs haem ligation during the assembly of c-type cytochromes. The focus herein is upon a newly discovered interaction of the CydDC complex with a haem cofactor; haem has recently been shown to modulate CydDC activity and structural modelling reveals a potential haem-binding site on the periplasmic surface of the complex. These findings have important implications for future investigations into the potential roles for the CydDC-bound haem in redox sensing and tolerance to nitric oxide (NO).
  • Yamashita, M. et al. (2014). Structure and Function of the Bacterial Heterodimeric ABC Transporter CydDC: Stimulation of the ATPase Activity by Thiol and Heme Compounds. The Journal of biological chemistry [Online] 289:23177-23188. Available at: http://dx.doi.org/10.1074/jbc.M114.590414.
    In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ABC- type cysteine/glutathione (GSH) transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two dimensional crystals of CydDC were analysed by electron cryomicrosopy and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ABC transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (ca. 100 nmol Pi/min/mg) that was stimulated approximately three-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC mediated transmembrane thiol transport.
  • Shepherd, M. et al. (2013). Structural and functional characterisation of ScsC, a periplasmic thioredoxin-like protein from Salmonella enterica serovar Typhimurium. Antioxidants and Redox Signaling [Online] 19:1494-1506. Available at: http://dx.doi.org/10.1089/ars.2012.4939.
    AIMS: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins has been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses.
    RESULTS: Herein, we describe the structural and biochemical characterisation of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of beta-sheets and alpha-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress.
    INNOVATION AND CONCLUSION: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity.
  • Tinajero-Trejo, M. and Shepherd, M. (2013). The globins of Campylobacter jejuni. Advances in microbial physiology [Online] 63:97-145. Available at: http://dx.doi.org/10.1016/B978-0-12-407693-8.00004-2.
    Campylobacter jejuni is a zoonotic Gram-negative bacterial pathogen that is exposed to reactive nitrogen species, such as nitric oxide, from a variety of sources. To combat the toxic effects of this nitrosative stress, C. jejuni upregulates a small regulon under the control of the transcriptional activator NssR, which positively regulates the expression of a single-domain globin protein (Cgb) and a truncated globin protein (Ctb). Cgb has previously been shown to detoxify nitric oxide, but the role of Ctb remains contentious. As C. jejuni is amenable to genetic manipulation, and its globin proteins are easily expressed and purified, a combination of mutagenesis, complementation, transcriptomics, spectroscopic characterisation and structural analyses has been used to probe the regulation, function and structure of Cgb and Ctb. This ability to study Cgb and Ctb with such a multi-pronged approach is a valuable asset, especially since only a small fraction of known globin proteins have been functionally characterised.
  • Taylor, S. et al. (2013). Measuring protein reduction potentials using 15N HSQC NMR spectroscopy. Chemical Communications [Online] 49:1847-1849. Available at: http://dx.doi.org/10.1039/c3cc38952a.
    NMR spectroscopy was used to measure reduction potentials of four redox proteins by following multiple 15N HSQC protein resonances across a titration series using mixtures of oxidised and reduced glutathione. Results for PDI a, PDI ab and DsbA agree with the literature and our result for ERp18 confirms this protein as an oxidoreductase of comparable or greater reducing strength than PDI a.
  • King, N. et al. (2012). Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance. BMC Microbiology [Online] 12. Available at: http://dx.doi.org/10.1186/1471-2180-12-8.
    The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.

    In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of ?-helices in a probable coiled-coil formation.

    Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated throughout the staphylococci.
  • Frey, A. et al. (2011). The single-domain globin of Vitreoscilla: augmentation of aerobic metabolism for biotechnological applications. Advances in Microbial Physiology [Online] 58:81-139. Available at: http://dx.doi.org/10.1016/B978-0-12-381043-4.00003-9.
    Extensive studies have revealed that large-scale, high-cell density bioreactor cultivations have significant impact on metabolic networks of oxygen-requiring production organisms. Oxygen transfer problems associated with fluid dynamics and inefficient mixing efficiencies result in oxygen gradients, which lead to reduced performance of the bioprocess, decreased product yields, and increased production costs. These problems can be partially alleviated by improving bioreactor configuration and setting, but significant improvements have been achieved by metabolic engineering methods, especially by heterologously expressing Vitreoscilla hemoglobin (VHb). Vast numbers of studies have been accumulating during the past 20 years showing the applicability of VHb to improve growth and product yields in a variety of industrially significant prokaryotic and eukaryotic hosts. The global view on the metabolism of globin-expressing Escherichia coli cells depicts increased energy generation, higher oxygen uptake rates, and a decrease in fermentative by-product excretion. Transcriptome and metabolic flux analysis clearly demonstrate the multidimensional influence of heterologous VHb on the expression of stationary phase-specific genes and on the regulation of cellular metabolic networks. The exact biochemical mechanisms by which VHb is able to improve the oxygen-limited growth remain poorly understood. The suggested mechanisms propose either the delivery of oxygen to the respiratory chain or the detoxification of reactive nitrogen species for the protection of cytochrome activity. The expression of VHb in E. coli bioreactor cultures is likely to assist bacterial growth through providing an increase in available intracellular oxygen, although to fully understand the exact role of VHb in vivo, further analysis will be required.
  • Burda, W. et al. (2011). Neutral metallated and meso-substituted porphyrins as antimicrobial agents against Gram-positive pathogens. European Journal of Clinical Microbiology & Infectious Diseases [Online] 31:327-335. Available at: http://dx.doi.org/10.1007/s10096-011-1314-y.
    Staphylococcus aureus is a bacterial pathogen that causes severe infections among humans. The increasing emergence of antibiotic resistance necessitates the development of new strategies to combat the spread of disease. One approach is photodynamic inactivation using porphyrin photosensitizers, which generate superoxide and other radicals in the presence of light, causing cell death via the oxidation of proteins and lipids. In this study, we analyzed a novel library of meso-substituted and metallated porphyrins for activity against multidrug-resistant S. aureus. From a library of 251 compounds, 51 showed antimicrobial activity, in three discrete classes of activity: those that functioned only in light, those that had toxicity only in darkness, and those that displayed activity regardless of illumination. We further demonstrated the broad-spectrum activity of these compounds against a variety of pathogens, including Bacillus anthracis, Enterococcus faecalis, and Escherichia coli. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) analyses of lead compounds (XPZ-263 and XPZ-271) revealed strong activity and killing towards methicillin-resistant S. aureus (MRSA) strains. An analysis of mutation frequencies revealed low incidences of resistance to lead compounds by E. coli and MRSA. Finally, an exploration of the underlying mechanism of action suggests that these compounds do not depend solely upon light-induced radical generation for toxicity, highlighting their potential for clinical applications.
  • Thomas, M. et al. (2011). Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on L-lactate. Environmental Microbiology [Online] 13:48-61. Available at: http://dx.doi.org/10.1111/j.1462-2920.2010.02307.x.
    Campylobacter jejuni, a major food-borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and L- and D-lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on L-lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, L-lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD-independent L-LDHs; a non-flavin iron-sulfur containing three subunit membrane-associated enzyme (Cj0075c-73c), and a flavin and iron-sulfur containing membrane-associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on L-lactate, as single mutants in each system grew as well as wild-type on this substrate, while a cj0075c cj1585c double mutant showed no L-lactate oxidase activity and did not utilize or grow on L-lactate; D-lactate-dependent growth was unaffected. Orthologues of Cj0075c-73c (LldEFG/LutABC) and Cj1585c (Dld-II) were recently shown to represent two novel families of L- and D-lactate oxidases; this is the first report of a bacterium where both enzymes are involved in L-lactate utilization only. The cj0075c-73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for L-lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use L- and D-lactate as both electron-donor and carbon source.
  • Smith, H. et al. (2011). The NO-responsive hemoglobins of Campylobacter jejuni: Concerted responses of two globins to NO and evidence in vitro for globin regulation by the transcription factor NssR. Nitric Oxide: Biology and Chemistry [Online] 25:234-41. Available at: http://dx.doi.org/10.1016/j.niox.2010.12.009.
    Campylobacter jejuni possesses NO-responsive and -detoxifying mechanisms to survive NO during transmission and pathogenesis. C. jejuni possesses two hemoglobins. The first (Cgb) is a single-domain (non-flavo)hemoglobin encoded by gene Cj1586 (cgb), mutation of which leads to hypersensitivity to S-nitrosoglutathione and NO. Transcription of cgb is induced by nitrosative stress and confers resistance to NO, presumably via a Cgb-catalyzed dioxygenase or denitrosylase reaction that converts NO and oxygen to nitrate. Expression of Cgb in response to NO is mediated via the positively-acting transcription factor NssR, which regulates expression of a small regulon that includes cgb and ctb (Cj0465c), the latter encoding the truncated hemoglobin, Ctb. The role of Ctb is unclear: it is not directly involved in NO detoxification but is implicated in oxygen delivery or metabolism. Here, we describe attempts to define a function for Ctb by examining the effects of a ctb mutation on the NO transcriptome and cgb gene expression during normoxia and hypoxia. Mutation of ctb does not elicit major compensatory transcriptomic changes but relatively minor changes in genes involved in intermediary metabolism, solute transport and signal transduction. We present and test the hypothesis that, by binding NO or O(2), Ctb dampens the response to NO under hypoxic conditions and limits cgb expression, perhaps because Cgb function (i.e. NO detoxification) requires O(2)-dependent chemistry. We report the purification of NssR and specific binding to the ctb promoter. GSNO does not affect the high affinity of NssR for the ctb promoter.
  • Arroyo Mañez, P. et al. (2011). Role of the distal hydrogen-bonding network in regulating oxygen affinity in the truncated hemoglobin III from Campylobacter jejuni. Biochemistry [Online] 50:3946-56. Available at: http://dx.doi.org/10.1021/bi101137n.
    Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.
  • Shepherd, M., Bernhardt, P. and Poole, R. (2011). Globin-mediated nitric oxide detoxification in the foodborne pathogenic bacterium Campylobacter jejuni proceeds via a dioxygenase or denitrosylase mechanism. Nitric Oxide: Biology and Chemistry [Online] 25:229-33. Available at: http://dx.doi.org/10.1016/j.niox.2010.12.006.
    Nitric oxide (NO()) is a toxin, but bacteria have evolved various strategies to detoxify this harmful radical to nitrate, the best known mechanism being the dioxygenase reaction of bacterial flavohaemoglobins. In addition, globins can form oxoferryl (Fe(IV)O) species through the reaction of the ferric haem with hydrogen peroxide: these species can also detoxify NO() to nitrite and nitrate. During infection, Campylobacter is exposed to both NO() and hydrogen peroxide. A question therefore arises: does Campylobacter jejuni utilize its single domain globin (Cgb) to detoxify NO() via the oxoferryl route, or via the more conventional dioxygenase or denitroxylase routes? The data herein demonstrate that the reaction between Cgb and hydrogen peroxide is much slower than for other globins, and subsequent reaction between the oxoferryl species and NO() is unfavourable. Furthermore, NO() may bind to Cgb in the oxyferrous, ferrous and ferric states. The ample opportunity for NO() to interact with ferrous and ferric Cgb, and the unfavourable reaction of ferric Cgb with hydrogen peroxide, suggests that NO() detoxification in C. jejuni proceeds via a dioxygenase or denitroxylase route requiring the haem iron to exist only in the Fe(II) or Fe(III) redox states.
  • Shepherd, M. et al. (2010). The single-domain globin from the pathogenic bacterium Campylobacter jejuni: novel D-helix conformation, proximal hydrogen bonding that influences ligand binding, and peroxidase-like redox properties. Journal of Biological Chemistry [Online] 285:12747-54. Available at: http://dx.doi.org/10.1074/jbc.M109.084509.
    The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.
  • Shepherd, M. et al. (2010). Compensations for diminished terminal oxidase activity in Escherichia coli: cytochrome bd-II-mediated respiration and glutamate metabolism. Journal of Biological Chemistry [Online] 285:18464-72. Available at: http://dx.doi.org/10.1074/jbc.M110.118448.
    Escherichia coli possesses cytochrome bo' (CyoABCDE), cytochrome bd-I (CydAB), and cytochrome bd-II (AppBC) quinol oxidases, all of which can catalyze the terminal step in the aerobic respiratory chain, the reduction of oxygen by ubiquinol. Although CydAB has a role in the generation of DeltapH, AppBC has been proposed to alleviate the accumulation of electrons in the quinone pool during respiratory stress via electroneutral ubiquinol oxidation. A cydB mutant strain exhibited lower respiration rates while maintaining a wild type growth rate. Transcriptomic analysis revealed a dramatic up-regulation of AppBC in the cydB strain, accompanied by the induction of genes involved in glutamate/gamma-aminobutyric acid (GABA) antiport, the GABA shunt, the glyoxylate shunt, respiration (including appBC), motility, and osmotic stress. Transcription factor modeling suggests that the underpinning regulation is largely controlled by H-NS, GadX, FlhDC, and AppY. The transcriptional adaptations imply that cydB cells contribute to the proton motive force via consumption of intracellular protons and glutamate/GABA antiport. Indeed, supplementation of culture medium with l-glutamate stimulates growth in a cydB strain. Phenotype analyses of the cydB strain confirm decreased motility and elevated acid resistance and also an elevated cytochrome d spectroscopic signal in cells grown at low pH. We propose a mechanism via which E. coli can compensate for the loss of cytochrome bd-I activity; cytochrome bd-II-mediated quinol oxidation prevents the accumulation of NADH, whereas GABA synthesis/antiport maintains the proton motive force for ATP production.
  • Mason, M. et al. (2009). Cytochrome bd confers nitric oxide resistance to Escherichia coli. Nature Chemical Biology [Online] 5:94-6. Available at: http://dx.doi.org/10.1038/nchembio.135.
    The aerobic respiratory chain of Escherichia coli has two terminal quinol oxidases: cytochrome bo and cytochrome bd. Cytochrome bd was thought to function solely to facilitate micro-aerobic respiration. However, it has recently been shown to be overexpressed under conditions of nitric oxide (NO) stress; we show here that cytochrome bd is crucial for protecting E. coli cells from NO-induced growth inhibition by virtue of its fast NO dissociation rate.
  • Shepherd, M. and Dailey, H. (2009). Peroxidase activity of cytochrome c facilitates the protoporphyrinogen oxidase reaction. Cellular and Molecular Biology [Online] 55:6-14. Available at: http://dx.doi.org/10.1170/T831.
    Protoporphyrinogen oxidase (PPO) catalyzes the penultimate reaction in heme biosynthesis. The 'oxygen dependent' form of this enzyme can utilize three molecules of oxygen as electron acceptors in the reaction. In the current study, the ability of cytochrome c to serve as an electron acceptor for PPO was examined. Cytochrome c was found to enhance the catalytic rate of Drosophila melanogaster PPO under reduced oxygen conditions, and cytochrome c became reduced during PPO catalysis. Further kinetic analysis under anaerobic conditions revealed that hydrogen peroxide, a byproduct of the PPO reaction, is required for this rate enhancement to occur. This suggests that the generation of free radicals via the peroxidase activity of cytochrome c plays a part in this rate enhancement, rather than cytochrome c acting as an electron acceptor for the PPO reaction. Given the abundance of cytochrome c in the intermembrane space of mitochondria, the cellular location of PPO, this process may potentially impact on the synthesis of heme in vivo particularly in conditions of low oxygen or hypoxia.
  • Mason, M. et al. (2008). A quantitative approach to nitric oxide inhibition of terminal oxidases of the respiratory chain. Methods in Enzymology [Online] 437:135-159. Available at: http://dx.doi.org/10.1016/S0076-6879(07)37008-0.
    Inhibition of terminal respiratory oxidases by nitric oxide (NO) plays important physiological roles in signaling and host defense. Using a bacterial quinol oxidase and mitochondrial cytochrome c oxidase, this chapter describes simple polarographic methods to quantify the kinetic characteristics of inhibition by NO. This chapter points out the inherent pitfalls of both experimental design and data analysis and compares alternative methods. Additionally, it describes a system designed to acquire polarographic and spectral data simultaneously to permit identification of spectral intermediates under defined conditions.
  • Shepherd, M., Heath, M. and Poole, R. (2007). NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein. Biochemistry [Online] 46:5030-5037. Available at: http://dx.doi.org/10.1021/bi700183u.
    NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.
  • Shepherd, M., Dailey, T. and Dailey, H. (2006). A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases. Biochemical Journal [Online] 397:47-52. Available at: http://dx.doi.org/10.1042/BJ20051967.
    Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.
  • Shepherd, M. and Dailey, H. (2005). A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation. Analytical Biochemistry 344:115-21.
    A continuous spectrofluorimetric assay for protoporphyrinogen oxidase (PPO, EC activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent Km (Kapp) values for protoporphyrinogen IX were measured as 3.8+/-0.3, 3.6+/-0.5, and 1.0+/-0.1 microM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The Ki for acifluorfen, a diphenylether herbicide, was measured as 0.53 microM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h-1/mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min-1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.
  • Shepherd, M., McLean, S. and Hunter, C. (2005). Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis. FEBS Journal [Online] 272:4532-4539. Available at: http://dx.doi.org/10.1111/j.1742-4658.2005.04873.x.
    Purified recombinant proteins from Synechocystis PCC6803 were used to show that the magnesium chelatase ChlH subunit stimulates magnesium protoporphyrin methyltransferase (ChlM) activity. Steady-state kinetics demonstrate that ChlH does not significantly alter the K(m) for the tetrapyrrole substrate. However, quenched-flow analysis reveals that ChlH dramatically accelerates the formation and breakdown of an intermediate in the catalytic cycle of ChlM. In light of the profound effect that ChlH has on the methyltransferase catalytic intermediate, the pre steady-state analysis in the current study suggests that ChlH is directly involved in the reaction chemistry. The kinetic coupling between the chelatase and methyltransferase has important implications for regulation of chlorophyll biosynthesis and for the availability of magnesium protoporphyrin for plastid-to-nucleus signalling.
  • Shepherd, M. and Hunter, C. (2004). Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase. Biochemical Journal [Online] 382:1009-13. Available at: http://dx.doi.org/10.1042/BJ20040661.
    Magnesium protoporphyrin IX methyltransferase (ChlM), an enzyme in the chlorophyll biosynthetic pathway, catalyses the transfer of a methyl group to magnesium protoporphyrin IX (MgP) to form magnesium protoporphyrin IX monomethyl ester (MgPME). S-Adenosyl-L-methionine is the other substrate, from which a methyl group is transferred to the propionate group on ring C of the porphyrin macrocycle. Stopped-flow techniques were used to characterize the binding of porphyrin substrate to ChlM from Synechocystis PCC6803 by monitoring tryptophan fluorescence quenching on a millisecond timescale. We concluded that a rapid binding step is preceded by a slower isomerization of the enzyme. Quenched-flow techniques have been employed to characterize subsequent partial reactions in the catalytic mechanism. A lag phase has been identified that has been attributed to the formation of an intermediate. Our results provide a greater understanding of this catalytic process which controls the relative concentrations of MgP and MgPME, both of which are implicated in signalling between the plastid and nucleus in plants.
  • Shepherd, M., Reid, J. and Hunter, C. (2003). Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803. Biochemical Journal [Online] 371:351-60. Available at: http://dx.doi.org/10.1042/BJ20021394.
    Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C(6) propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases. Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP. Initial rate studies showed that the reaction proceeds via a ternary complex. Product (S-adenosyl-L-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further. SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD. This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme or a ChlM-MgD complex. Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.

Book section

  • Burda, W., Shaw, L. and Shepherd, M. (2012). Porphyrins: Properties and Applications. in: Kaibara, A. and Matsumara, G. eds. Handbook of Chemistry, Biochemistry and Biology: New Frontiers. Nova Publishers, pp. 429-438.
  • Poole, R. and Shepherd, M. (2012). Bacterial Globins. in: Roberts, G. C. K. ed. Encyclopedia of Biophysics. EBSA Publishers, pp. 156-160.
  • Shepherd, M. and Poole, R. (2012). Bacterial Respiratory Chains. in: Roberts, G. C. K. ed. Encyclopedia of Biophysics. EBSA Publishers, pp. 172-177.
  • Shepherd, M. and Dailey, H. (2004). Porphyrin Metabolism. in: Lennarz, W. J. and Lane, M. D. eds. Encyclopedia of Biological Chemistry. Elsevier Publications, pp. 415-419.
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