Professor
Department of Biology
Krieger School of Arts & Sciences

joel@jhu.edu

235 Mudd Hall
3400 N. Charles Street
Baltimore, MD 21218

Office: 410-516-0176
Lab: 410-516-0177

My lab studies the structural biology of bacterial conjugation. Conjugation is a method of DNA transfer that can occur between even distantly related bacterial species, facilitating the dissemination of drug resistance and virulence factors throughout bacterial populations. By exploiting a variety of biophysical and biochemical techniques including X-ray crystallography, electron microscopy, and single-molecule fluorescence spectroscopy, we intend to describe the various steps in this complex biological process in molecular and structural terms, attaining atomic resolution wherever possible.

During bacterial conjugation, plasmid-encoded Tra proteins direct transfer of DNA, in single-stranded form, from a donor to a recipient cell. For F plasmid of E. coli, one of the better-characterized conjugative plasmids, the process initially involves expression of pili by the donor bacterium. Recipient bacteria can adhere to the pili, and through retraction of a pilus, the donor and recipient come into contact, eventually forming a stable "mating pair". Plasmid DNA in the donor is nicked, unwound, and separated into single strands. One strand is transferred to the site of contact between the cells and transported across the membranes into the recipient. The DNA in the recipient is circularized, and complementary DNA strands are synthesized in both donor and recipient. While the F plasmid tra genes have all been sequenced and the system thoroughly studied on the genetic level, considerably less is known about the biochemical or structural bases of the activity of the Tra proteins.

Our current focus is the DNA nicking and initiation of transfer of F plasmid. The TraI protein is central to both of these steps.  TraI possesses both DNA nucleolytic and DNA helicase activities. TraI does not act alone, however, with its optimal nicking activity requiring two accessory proteins. These are the F plasmid-encoded TraY, and the host-encoded integration host factor (IHF), both of which bind to DNA sequences proximal to the TraI nicking site. Together, the three proteins form a complex called the relaxosome.  The TraI DNA cleavage activity is actually a transesterification reaction in which a covalent linkage between TraI and the DNA is formed.  Following DNA cleavage, unwinding of the plasmid by TraI is delayed until a signal indicating formation of a stable mating complex is received. Responding to this signal, the TraI molecule is converted from an apparently quiescent state to a functioning helicase. We are attempting to answer several questions. How stable is the relaxosome in vivo?  Are all three proteins essential to relaxosome formation?  Does relaxosome formation require both TraI activities? Is TraI a pilot protein, leading the cleaved plasmid strand into the recipient?  If so, what is the physical state of TraI during transport and what characteristics of TraI are required to permit transport? If TraI is transported and must be denatured prior to transport, what denatures the TraI and what regulates that process?

We are examining the in vitro functions of the proteins, characterizing their interactions, and attempting to gather both low and high resolution structural data. The data on protein function will be analyzed with reference to the structural data, and these data will be used in turn to plan experiments to further characterize the proteins and to test and refine proposed models of function. By careful in vitro examination of these (and eventually additional) Tra proteins, we will piece together a detailed model of how this complex biological process proceeds in vivo.

Selected Publications
Williams, S.L., and J.F. Schildbach (2007) TraY and integration host factor oriT binding sites and F conjugal transfer: Sequence variations, but not altered spacing, are tolerated. J. Bacteriol. 189:3813-3823.

Williams, S.L., and J.F. Schildbach. (2006) Examination of an inverted repeat within the F factor origin of transfer: Context dependence of F TraI relaxase DNA specificity. Nucleic Acids Res. 34:426-435.

Larkin, C., S. Datta, M.J. Harley, B.J. Anderson, A. Ebie, V. Hargreaves, and J.F. Schildbach. (2005) Inter- and intramolecular determinants of the specificity of single-stranded binding and cleavage by the F factor relaxase. Structure (Camb) 13:1533-1544.

Stern, J.C., B.J. Anderson, T.J. Owens, and J.F. Schildbach. (2004) Energetics of the sequence-specific binding of single-stranded DNA by the F factor relaxase domain. J. Biol. Chem. 279:29155-29159.

Datta, S., C. Larkin, and J.F. Schildbach. (2003) Structural insights into single-stranded DNA binding and cleavage by F factor TraI. Structure 11:1369-1379.

Harley, M. J., and J.F. Schildbach. (2003) Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100. Proc. Natl. Acad. Sci. USA 100:11243-11248.

Larkin, C., S. Datta, A. Nezami, J.A. Dohm, and J.F. Schildbach. (2003) Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI. Acta Crystallogr. D. 59:1514-1516.

Miller, D.L., and J.F. Schildbach. (2003) Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM. J. Biol. Chem. 278:10400-10407.

Street, L.M., M.J., Harley, J.C. Stern, D.L. Miller, C. Larkin, S.L. Williams, J.A. Dohm, M.E. Rodgers, and J.F. Schildbach. (2003) Subdomain organization and catalytic residues of the F factor TraI relaxase domain. Biochimi. Biophys. Acta 1646:86-99.

Harley, M.J., D. Toptygin, T. Troxler, and J.F. Schildbach. (2002) R150A mutant of F TraI relaxase domain: Reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide. Biochemistry 41:6460-6468.

Lum, P. L., M. Rodgers, and J.F. Schildbach. (2002) TraY DNA recognition of its two F factor binding sites. J. Mol. Biol. 321:563-574.