Joel Schildbach

Joel Schildbach

netbet casino app

Contact Information

NetBet live casinoStructural biology of bacterial conjugation

netbet casino reviewJoel Schildbach is currently the program director for the Division of Graduate Education at the National Science Foundation.

He graduated with a BA from Oregon State University and a PhD from Harvard University, and then did his postdoctoral work at the Massachusetts Institute of Technology before arriving at Johns Hopkins University in 1996.

Bacterial conjugation, transfer of a plasmid from one bacterium to another, accelerates diversification of prokaryotic genomes and serves as perhaps the most important conduit for transfer of genes encoding antibiotic resistance and virulence factors. Yet despite its biological importance, our knowledge of conjugation at the molecular level is incomplete. By exploiting a variety of biophysical and biochemical techniques including X-ray crystallography, NMR, fluorescence spectroscopy, and in vivo fluorescence microscopy, we intend to describe the various steps in this complex biological process, attaining atomic resolution wherever possible. The information we obtain will enable us to better understand the function, organization, netbet online sports bettingregulation and structures of the macromolecular assemblies that carry out this process.

During bacterial conjugation, plasmid-encoded Tra proteins direct transfer of plasmid DNA, in single-stranded form, from a donor to a recipient cell. For F plasmid of E. coli, the first identified of the conjugative plasmids, the process initially involves expression of pili by the donor bacterium. The pili adhere to recipient bacteria 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 basis of the activity of the Tra proteins.

Proposed role of TraI in F plasmid transfer

Our current focus is the DNA nicking and initiation of transfer of F plasmid. The TraI protein and its two activities, as an ssDNA transesterase or "relaxase" and as a helicase, are central to the process. TraI does not act alone: its optimal activity against ssDNA comes when it participates in a complex with two proteins, the F-encoded TraY, and the host-encoded integration host factor (IHF). TraY and IHF both bind DNA sequences proximal to the TraI nicking site. Following DNA nicking (and cross-linking of TraI to ssDNA), ssDNA transfer is delayed until a signal indicating formation of a stable mating complex is received. Responding to this signal, the TraI molecule, in an effort that requires its helicase activity, is transported into the recipient and circularizes the transferred plasmid by reversing the initial transesterification reaction. 

Among the topics we are investigating:

ssDNA Recognition by TraI
We have shown that the TraI relaxase domain binds ssDNA with exquisite sequence specificity, and havedescribed the structural basis for this netbet casino reviewspecificity. The TraI helicase also binds ssDNA. Recently we discovered that these two sites, which can bind ssDNA with similar affinities, compete for ssDNA, with binding to one site preventing binding to the second. We believe that this characteristic plays an essential role in regulating the activities of TraI during transfer. Using a combination of NMR, X-ray crystallography, small-angle X-ray scattering and small-angle neutron scattering, we are working toward a structural model of the intact TraI protein. Using biochemical approaches, we are characterizing the helicase binding site and searching for the basis of the competition between binding sites.

The F TraI relaxase domain structure (top) reveals that ssDNA sequence specificity can be partially attributed to bases docking into pockets in the binding cleft (bottom).

In vivo imaging of the transfer process
We can measure the efficiency of the transfer process, and thus the effect of Tra protein variants, by assessing the frequency with which a genetic marker (for example, an antibiotic netbet online sports bettingresistance gene) moves from one bacterial strain to another. The transfer process, however, is complex, involving many steps. We can therefore determine that transfer has been impaired, but we have a much harder time determining at which step transfer is impaired. To counter this shortcoming, we are collaborating with the lab of Professor Jie Xiao to refine methods of observing the proteins and process of transfer as conjugation occurs.

Conjugative plasmids in virulence of bacterial pathogens
Although plasmids are sometimes mistakenly thought of as bacterial excess baggage, plasmids have co-evolved with their harboring strain, making the plasmid an integral part of the organism. There are reports that pathogenic bacterial strains harboring F-like plasmids can lose virulence when certain of the F-like genes are disrupted. What is the basis of this loss of virulence?  We are starting to explore the interactions between host and plasmid that can affect the pathogen.

See publications on ResearchGate.