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Gene transfer mediated by virus-like particles; light-driven reactions in photosynthesis proteins; applications of photosynthesis proteins to solar energy.
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
The Rhodobacter sphaeroides photosynthetic reaction centre (RC) is a pigment-protein complex that efficiently captures and converts photon energy into a charge-separated state. Given the conversion efficiency and the high electric potential of the electron, the major focus of my project was to deliver/extract electrons to/from various cofactors along the charge-separation pathway in the RC, including the special pair of bacteriochlorophylls (P), the bacteriopheophytin (HA), the primary quinone (QA), and the secondary quinone (QB). An over-expression system was created to produce RCs, using the R. sphaeroides RCx strain, pIND4 plasmid, a modified culture medium, and changes to growth conditions. These changes resulted in a 35-fold increase in protein levels compared to the previous system. To extract electrons from the quinone region of the RC, this region was made more accessible to the solvent by deleting portions of the H subunit cytoplasmic globular domain. The results indicated that the truncated RC mutants assembled stably and thereby reduced the electron transfer distance between the quinone and an external electron acceptor. Photochronoamperometry measurements on mutant RCs designed to test the feasibility of delivering electrons from an electrode to P showed photocurrent generation and direction that were consistent with the binding of the RC P-side to the electrode surface. Similar experiments on the feasibility of extracting electrons from HA, QA and QB, for delivery to highly ordered pyrolytic graphite (HOPG) or gold electrodes, also showed photocurrent generation and direction consistent with the binding of the RC HA-side or Q-side to the electrode surface. Finally, the thermal stability of complexes was studied by in vivo addition of light harvesting complex 1 (LH1) from the thermophile Thermochromatium tepidum to the RC. A hybrid core complex consisting of an R. sphaeroides RC surrounded by T. tepidum TLH1 conferred greater tolerance to thermal energies, compared to the analogous R. sphaeroides RC-LH1 core complex, at temperatures up to 70 °C. The combination of these results show that, in principle, the RC can be modified to extract electrons at different energy levels, or band gaps, with possible applications in heat-stabile biohybrid solar cell technologies.
The Rhodobacter capsulatus gene transfer agent (RcGTA) is a phage-like particle that mediates horizontal gene transfer between R. capsulatus strains, and its production is regulated by several bacterial systems, including quorum sensing and the CckA-ChpT-CtrA phosphorelay. This thesis presents evidence that RcGTA is released from cells by cell lysis, that lysis is modulated by the concentration of inorganic phosphate in the growth medium and that lysis requires an endolysin and holin gene. The expression of the lysis genes is regulated by the histidine kinase CckA, the phosphotransferase ChpT and the response regulator CtrA, and requires phosphorylation of CtrA. The endolysin and holin were characterized by expression in E. coli. High resolution electron microscopy images of affinity-purified RcGTA confirmed that RcGTA contains tail fibers and head spikes, and newly revealed the presence of a baseplate-like structure. RcGTA was found to undergo a maturation process similar to that of phages, and this maturation was regulated by the CckA-ChpT-CtrA phosphorelay. Cells lacking CckA produced tail-less particles containing DNA and polytube structures. During particle assembly spikes are attached to the head of RcGTA, and spike formation required ghsA and ghsB, which appear to be co-transcribed and regulated by CckA-ChpT-CtrA and quorum sensing. Spikes were required for efficient binding of RcGTA to the R. capsulatus capsular polysaccharide. Two new regulators of RcGTA, ClpX and DivL, were identified. ClpX was required for transduction, and capsid formation in cells lacking ClpX appeared to halt RcGTA production prior to DNA packaging. DivL appeared to be involved in regulating the CckA kinase activity; however a loss of DivL resulted in opposite phenotypes for an RcGTA overproducer and a wild type strain. Additionally, RcGTA production was found to be stimulated by temporary depletion of amino acids, but did not require the (p)ppGpp-mediated stringent response or a homologue of the general stress response sigma factor EcfG.
Gene transfer agents (GTAs) are agents of genetic exchange that resemble small tailed DNA bacteriophages and transfer random segments of the producing cell's genome to recipient cells. The canonical GTA is produced by the α-proteobacterium Rhodobacter capsulatus, hereafter referred to as RcGTA. The RcGTA packages ~4 kb segments of genomic DNA, and is produced and released by the lysis of a sub-population of donor cells in the stationary phase of growth. The primary structural gene cluster is a ~ 15 kb genomic region. Production and release of RcGTA is regulated by several host systems, including the GtaI quorum-sensing system, and the CckA/ChpT/CtrA putative phosphorelay system. Prior to this work, studies on RcGTA focused primarily on aspects involved in the production of RcGTA particles, such as gene regulation, DNA packaging, and biological functionality. However essentially nothing was known about how RcGTA delivers DNA to recipient cells. Herein, several key aspects of the capability of a cell to receive an RcGTA carried genetic marker, defined as RcGTA recipient capability, are delineated. Initial studies on the GtaR/I quorum-sensing system showed that gtaR/I are co-transcribed, and indirectly regulate not only transcription of the RcGTA gene cluster, but also RcGTA recipient capability. Part of this quorum-sensing effect was attributed to regulation of capsular polysaccharide production, which was determined to be involved in RcGTA adsorption to cells. Additionally, it was found that CtrA is essential for, and a regulator of several genes required for RcGTA recipient capability. CtrA was found to regulate a set of natural competence genes involved in DNA entry into the cell and in RecA-mediated homologous recombination. These genes, DprA, ComM, ComEC, and ComF, are all essential for RcGTA recipient capability, and analyses of the encoded proteins were used to propose a pathway for acquisition of RcGTA-borne DNA. These findings indicate that the RcGTA horizontal gene transfer mechanism is a combination of two fundamentally different horizontal gene transfer (HGT) mechanisms, transduction and transformation, generating a very efficient mode of HGT.
The photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides is a pigment-protein complex in which electron transfer reactions efficiently capture solar energy in the form of charge separation. Embedded in this protein are two molecules of bacteriopheophytin (BPhe), one of which mediates electron transfer (HA). Efficient electron transfer through HA, as well as other cofactors of the RC, relies on both the spatial arrangement of the cofactors in the protein scaffold, as well as favorable interactions between the cofactors and their surrounding protein components. In this work, the interplay between the HA cofactor and its protein environment are explored. Specifically, a leucine residue whose side chain projects orthogonally to the plane of the HA macrocycle was changed by site-directed mutagenesis, creating a series of RC mutants with different side chains at this position (termed (M)214). The results derived from this work reveal that: (i) (M)214 plays a role in the pigment selectivity of the HA binding pocket; (ii) the volume of the (M)214 side chain is important for fast forward electron transfer; and (iii) the (M)214 residue affects the configuration of a nearby bacteriochlorophyll pigment. In a subsequent chapter of this work, I describe the effects of HA coordination state in RCs that assemble exclusively with zinc-bacteriochlorophyll in place of bacteriochlorophyll and BPhe. My results reveal that the coordination state of the HA Zn²⁺ metal plays a role in determining the yield of charge separation within the protein. In addition, it was found that these so-called Zn-RCs do not assemble with a full occupancy of a cofactor. Collectively, the results from this work serve to increase our basic understanding of the protein-cofactor interplay within photosynthetic systems.
The work in this thesis reports the first discovery of flagellum-independent motility in Rhodobacter capsulatus, a purple photosynthetic bacterium. Furthermore, while aqueous swimming motility using a flagellum had been documented, the occurrence of movement over solid and semi-solid substrates had not been reported in R. capsulatus. This motility was found to be affected by the physical and chemical composition of the translocation surface. While motility was reduced under anaerobic (dark) conditions, it did not require oxygen. Cells appeared to respond to multiple stimuli, and were able to move both as coordinated masses and individual cells. Coordinated movements did not require any of the known cell-to-cell communication mechanisms. Movements were influenced by light such that cells usually moved toward a light source over a broad region of the visible spectrum, and this movement appeared to be a genuine phototaxis. A direct linkage between photoresponsive movement and photosynthesis was ruled out, because the photosynthetic reaction center was not required for movement toward white light. Photoresponsive movement occurred independently of the photoactive yellow protein, but appeared to require the bacteriochlorophyll and/or carotenoid pigments. Motility was mediated by flagellum-dependent and flagellum-independent contributions. Flagellum-dependent contributions were responsible for dispersive semi-random movements while flagellum-independent contributions resulted in linear, directed movements. Analysis of several strains indicated that flagellum-independent motility is widespread throughout R. capsulatus. This motility appears to be mediated by a gliding mechanism, perhaps involving the deposition of exopolysaccharide to achieve coordinated cell taxis.
Bacteria are found in almost all conceivable environments, and some species can survive many different conditions. The ability to detect environmental conditions and respond with appropriate changes to gene expression is essential to survival. Bacteria sometimes express genes involved in horizontal gene transfer when encountering a stressful environment. Horizontal gene transfer has an important role in the evolution of prokaryotic genomes. Rhodobacter capsulatus produces a mediator of horizontal gene transfer called the gene transfer agent (GTA). The R. capsulatus GTA is a bacteriophage-like particle that transfers ~4 kb of double stranded genomic DNA using a transduction-like mechanism. Previously, two proteins encoded outside the GTA gene cluster, GtaI and CtrA, were found to regulate GTA expression. GtaI and GtaR are LuxI-type and LuxR-type quorum sensing proteins, respectively. CtrA and CckA are homologues of the response regulator and sensor kinase, respectively, of the Caulobacter crescentus CtrA/CckA signal transduction system. In this thesis, I studied the interactions between these regulatory proteins, environmental conditions and GTA in R. capsulatus. I found that growth conditions had opposite effects on GTA and ctrA expression, but no effect on gtaR expression, and phosphate limitation decreased expression of ctrA. Knockout experiments revealed that GtaI and GtaR affect ctrA, gtaR and GTA expression. Results from GtaR-DNA binding experiments were consistent with a model in which GtaR directly regulates its own expression but indirectly regulates ctrA and GTA expression. These studies also identified GtaR binding sequences. I found that in R. capsulatus CtrA did not regulate its own transcription, contrary to what occurs in C. crescentus. My research also showed that GTA expression was affected by at least one other unidentified system. Promoter deletion studies of ctrA, gtaR and GTA genes identified sequences that may be involved in GtaI-, GtaR-, CtrA-, and/or growth condition-based regulation. Overall, these studies contribute to the understanding of how bacteria detect multiple environmental signals and respond with changes to gene expression.
Porphyrins, for example heme and chlorophyll, are vital to biological processes such as respiration and photosynthesis. Both cofactors are synthesized through a common pathway to protoporphyrin IX (PPIX) which then branches: Fe²⁺ chelation into the macrocycle by ferrochelatase results in heme formation; by contrast, Mg²⁺ addition by Mg-chelatase commits the porphyrin to (bacterio)chlorophyll synthesis. The purple bacterium Rhodobacter sphaeroides is a model for bacteriochlorophyll a (BChl) biosynthesis and type-2 reaction center (RC) structure and function. While studying RC protein assembly it was discovered that a bchD (Mg-chelatase) mutant did not produce BChl as wild-type (wt) cells do, but instead produced small quantities of an alternative BChl in which Mg²⁺ was substituted by Zn²⁺. Zn-BChl α has been found in only one other organism before, the related acidophilic purple phototrophic bacterium Acidiphilium rubrum. The overall objectives of this thesis were two-fold: (1) to elucidate the Zn-BChl biosynthetic pathway; and (2) to utilize this biosynthesis as a tool to probe aspects of photosynthetic apparatus function.The biosynthetic pathway of Zn-BChl in the bchD mutant was found to begin at ferrochelatase, which efficiently chelated Zn²⁺ into PPIX. The resultant Zn-PPIX was utilized by the BChl-biosynthetic pathway, with metabolites early in the pathway accumulating, but with low Zn-BChl levels. Two novel intermediates I described, protoporphyrin IX monomethyl ester and divinyl-protochlorophyllide, contained Zn²⁺ instead of Mg²⁺. The demonstrated Zn-BChl biosynthetic pathway is a new way to make BChl and facilitates the further engineering of alternate forms of (Zn-)BChl. The RC in the bchD mutant (Zn-RC) was found to bind six Zn-BChls, instead of the four Mg-BChls and two bacteriopheophytins of the WT-RC. Spectroscopic examination showed that electron transfer (ET) in the Zn-RC occurs at approximately the same rate as in the WT-RC, despite substitution of Zn-BChl for bacteriopheophytin α. We showed preservation of ET was due to the unusual tetracoordination state of the Zn-BChls in the bacteriopheophytin site. This discovery allows refinement of ET rules within pigment-protein complexes by showing that the coordination state and conformation of cofactors can have an equally important role as the protein.
Master's Student Supervision (2010 - 2018)
The Rhodobacer capsulatus gene transfer agent (RcGTA) is a phage-like particle capable of packaging, and transferring, ~4 kbp fragments of DNA between different strains of R. capsulatus. This genetic transfer is dependent upon a variety of factors, including the regulation of transcription of the main structural gene cluster, particle maturation, release, and uptake (RcGTA recipient capability) by other, ‘RcGTA competent’ cells. These processes have been previously demonstrated to be regulated by the CckA-ChpT-CtrA phosphorelay pathway. I have created three CckA site-directed mutants thought to be involved in mediating kinase, phosphatase, or cyclic-di-GMP binding activities of the CckA protein. My results provide strong evidence that these three activities play roles in regulating the transcription, maturation, and RcGTA ‘competency’ of these cells. My thesis also provides evidence that the ChpT, and DivL regulatory proteins play a role in the regulation of RcGTA recipient capability. I further demonstrate that cell growth and morphology are not noticeably affected by mutations in CckA activity, and that differences in RcGTA recipient capability are not due to differences in the ability of RcGTA particles to bind, or adsorb, to cells. Overall, my thesis provides novel insights in to how RcGTA production and recipient capability are regulated, furthering our understanding of how this novel horizontal gene transfer mechanism is regulated.
Rhodobacter capsulatus is a metabolically versatile α-proteobacterium that produces abacteriophage-like particle called the gene transfer agent (RcGTA) that is capable of mediatinghorizontal gene transfer. RcGTA particles transfer random 4.5 kb fragments of genomic DNAthat integrate into recipient genomes by allelic replacement. This thesis addresses certainenvironmental conditions, in particular carbon limitation and the presence of subinhibitoryconcentrations of antibiotics, that influence gene transfer by RcGTA. A new transduction assaywas developed to test the effects of various substances on gene transfer. Using this transductionassay, both carbon limitation and low levels of DNA gyrase inhibitors were found to increase thefrequency of gene transfer, although by different mechanisms. Carbon limitation caused anincrease in production and release of RcGTA. This effect was a general response to carbonlimitation, and was independent of carbon source. Gyrase inhibitors, on the other hand, did notinfluence production or release of RcGTA and instead were thought to act on the recipient cellsvia DNA gyrase. GyrB overexpression constructs were made in order to confer resistance tonovobiocin. The presence of these constructs negated the novobiocin-mediated increase in genetransfer. The results of this thesis suggest that certain antibiotics as well as carbon limitationaffect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange innatural ecosystems.