…Current Projects

The specific aims of the project are:

1. To assess the tempo and mechanisms of plasmid evolution during vertical transmission in a single host as compared to vertical and horizontal transmission among phylogenetically distinct hosts in the presence of selective pressure;
2. To characterize and compare the genetic and phenotypic changes that occur during such experimental plasmid evolution; and
3. To test the ability of various algorithms to accurately reconstruct the true phylogenies of independently evolved plasmids and specific genes.

Since plasmids with a broad host-range play the most important role in spread of resistance between phylogenetically distinct hosts, we chose the 64.5 kb broad-host-range IncP-1beta plasmid pB10 as a model plasmid for our studies (see diagram below). This plasmid was isolated from a wastewater treatment plant in Germany, see Dröege et al.2000 [pdf], and passage in lab cultures since its isolation had been kept to a minimum. In collaboration with the group of Professor Pühler at the University of Bielefeld, Germany, its complete DNA sequence has been obtained and annotated. We are currently examining how the plasmid evolved in E. coli over 500 generations of growth. Phenotypic changes are characterized by examining the effect of the evolved plasmid on host fitness, and by assessing differences in the stability and broad host range characteristics of the evolved and ancestral plasmids. Genetic changes that may account for the observed phenotypic differences are identified by comparing the plasmid expression profiles, by sequencing various regions of the evolved plasmids involved in replication, maintenance and transfer, and by characterizing macroscale variations. Several of our analyses are made possible thanks to the Molecular Biology Core Facility at the University of Idaho.

Lab members working on this project are Stacey Poler (scientific aide), Leen De Gelder (graduate student), Holger Heuer (postdoctoral researcher), and Monica Flory and Randal Fox (graduate student). This project is in collaboration with Dr. Larry Forney, Dr. Paul Joyce, and Dr. Steve Krone from the University of Idaho, and with Dr. C.M. Thomas from the University of Birmingham, UK.
For more details on this project, go to the individual lab members‘ information, or contact me, evatop [at] uidaho.edu.

Evo-X is a computer program used to analyze experimental evolution data. Evo-X uses likelihood methods for evaluation of models and estimation of mutation rates and selection coefficients of mutants arising in experimental evolution.

This program is associated with De Gelder et.al. (2004), wherein a mathematical model is introduced that aims to explain the evolutionary process of the loss of the tetracycline resistance operon from a plasmid contained in E. coli. This model is coupled with a statistical framework based on the maximum likelihood to estimate its parameters and to test the associated hypotheses.

From: Schlüter A., H. Heuer, R. Szczepanowski, L. J. Forney, C. M. Thomas, A. Pühler & E.M. Top. 2003.
The 64,508 bp IncP-1β antibiotic multiresistance plasmid pB10 isolated from a wastewater treatment plant provides evidence for recombination between members of different branches of the IncP-1β group.
Microbiology 149: 3139-3153.

To better define the gene pool shuttled among prokaryotes by broad-host-range plasmids and the effect that these genes have on the adaptive evolution of prokaryotes, we have initiated a large-scale project to determine the sequences of 100 BHR plasmids. This will add significant new data to the paltry plasmid sequence database that now exists, which is also skewed towards plasmids relevant to human infectious diseases. The plasmids sequenced are obtained from soil, water, and sewage sludge samples from around the globe. This effort is the first step toward our long-term goal to understand the nature and evolutionary history of self-transmissible BHR plasmids and their role in chromosomal gene exchange.

Last year the Department of Energy accepted our proposal to have 100 such plasmids sequenced by the Joint Genome Institute (JGI). The specific objectives of the research proposed here are: (i) to annotate the complete sequences of these 100 plasmids; (ii) to analyze the diversity and likely function of ‘accessory’ genes and gain clues to their origin; (iii) to examine the presence of plasmid sequences in bacterial chromosomes, and (iv) to assess the phylogenetic relatedness of genes found in plasmid ‘backbones’ to determine the evolutionary history of these plasmids.

We will also disseminate our work through Web delivery focused on middle school through college students, their teachers and advisors as well as the public. Some web features will include innovative information graphics, audio and video podcasts focused on “Issues in Science”, a frequently updated blog, and laboratory lesson examples to share with middle and high school teachers. This part of the work is done in collaboration with Arts & Design professors Frank Cronk and Jill Dacey, with the help of graphic design student Kiley Pheifer.

This project is funded by the Microbial Genome Sequencing program of the National Science Foundation (NSF).

If you are an undergraduate student from one of Idaho’s colleges, a high school student, and interested in working with us on this project, please contact Eva Top evatop [at] uidaho.edu. We also have a position for a postdoctoral scientist or Ph.D. student with experience in comparative genomics; contact us if you are interested and qualified!

Therefore we have started a joint theoretical and experimental investigation into the role of spatial structure in the spread and persistence of self-transmissible antibiotic resistance plasmids. The specific aims of this project are to construct 2-dimensional (2-D) and 3-dimensional (3-D) stochastic cellular automata (CA) models that can be used to accurately predict the spread and persistence of natural antibiotic resistance plasmids in bacterial colonies growing on agar surfaces and in biofilms. Experiments to optimize the 2-D model include monitoring plasmid transfer on agar plates, whereas biofilm studies will be performed to provide data for the construction of the 3-D model. See pictures below of red fluorescent cells growing on agar: They turn white when they lose their plasmid with inserted rfp gene.

In the laboratory of Dr. Eva Top (Biological Sciences), colonies of antibiotic resistant bacteria were grown on agar plates in the absence of antibiotics: Cells containing an antibiotic resistance plasmid are red because of a red fluorescent marker inserted on the plasmid. This plasmids is a circular DNA molecule that codes for resistance to four different drugs and mercury chloride. It can transfer between cells, and can also be lost from the cell when the cell divides. Red, plasmid-containing cells were inoculated in the center of the agar plate. As the colony grew, cells slowly (left) or rapidly (right) lost their plasmid and therefore turned white. The results show that while the drug resistance plasmid is very unstable in some hosts (right), it is rather stable in others (left) , indicating that the absence of drugs does not necessarily mean the loss of drug resistance in bacteria. The spatial structure of the colonies is of importance to understand and predict the spread and persistence of drug resistance genes in bacteria, and is being modeled by collaborator Dr. Stephen Krone (Mathematics) and his students.

Plasmid-bearing donor cells (red) and plasmid-free recipient cells (green) are growing in a biofilm, and conjugative plasmid transfer from donor to recipient results in plasmid-bearing recipients (called transconjugants, yellow).