Top Lab: Research
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Current Projects | Views of the
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Current Projects:
The Genetic Diversity of
Broad-host-Range Plasmids in Bacteria
The genomes of prokaryotic species are now known to be genetic
mosaics that have evolved over time through horizontal as well as
vertical inheritance of genetic material. Horizontally inherited genes
constitute a ‘virtual genome’ that is shared among a spectrum of
prokaryotic species. Broad-host-range (BHR) plasmids are an important
means by which genes are horizontally exchanged. The wholesale
acquisition of genes constitutes a clever way to acquire phenotypic
traits such as antibiotic and heavy metal resistance that can confer
important advantages to a host, and genetic material that can be
reshuffled or integrated into the host chromosome.
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.
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@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!
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Evolution of
Broad Host Range Antibiotic Resistance Plasmids
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| The main research project in the lab is part of an
NIH-funded COBRE grant that established the
Center for Research on
Evolutionary Processes (CRePE) at the University of Idaho. The
objective of this research is to discern patterns of plasmid evolution
in bacteria through experimental evolution studies. Among the traits
encoded by various plasmids are those that encode resistance to
antibiotics. The spread of antibiotic resistance among bacterial
species decreases their effectiveness for the treatment of infectious
diseases. In addition, plasmids encode functions that are essential
for their replication, maintenance and transfer, and these determine
the host range of these genetic elements. The specific aims of the
project are:
- 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;
- To characterize and compare the genetic and phenotypic changes
that occur during such experimental plasmid evolution; and
- 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). This plasmid was isolated from
a wastewater treatment plant in Germany (Droege et al.2000,
view
PDF (requires
Acrobat Reader), and passage in lab cultures since its
isolation had been kept to a minimum. In collaboration with the group
of
Professor Puehler at the University of Bielefeld, 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
(undergraduate students). This project is in collaboration with
Dr. Larry Forney,
Dr.
Paul Joyce, and
Dr.
Steve Krone from the U of I, and with
Dr. C.M. Thomas from the University of Birmingham, UK. For more details on this project, go
to the individual lab members'
pages, or contact me (evatop@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.
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Effect of the bacterial host on plasmid
transfer and stability
Although the host cell
is the plasmid’s primary environment, possible effects of the
bacterial host on plasmid properties have not been investigated. This
project examined to what extent the bacterial host influences certain
plasmid characteristics such as the plasmid transfer range in a
microbial community and plasmid stability. In general, this work
pioneered the notion that the bacterial host can have a pronounced
effect on the properties of the plasmid it carries. Therefore, a
plasmid cannot be regarded as a fixed entity entirely defined by the
genes it encodes, but has to be thought of as having flexible
properties in different hosts. This work was performed by
Leen De
Gelder as
part of her PhD thesis work.
To investigate whether
the host range of a BHR multiresistance plasmid within an activated
sludge microbial community depends on the plasmid carrying donor
strain, separate conjugation experiments with three pB10::rfp
donors were carried out in replicate samples of fresh activated sludge
from a wastewater treatment plant. The phylogeny of 306
transconjugants was determined by partial 16S rRNA gene sequence
analysis. Statistical analysis showed that the phylogenetic
diversities of transconjugants obtained using the three donors were
significantly different. Our results indicate that the spectrum of
plasmid recipients can be strongly influenced by the plasmid
donor. This study may stimulate further research on the factors
that determine the host range of plasmids in microbial communities.
Supplementary material
associated with the publication about this work (De Gelder et al.,
2005,
Appl. Envir. Microb.)
To examine whether the
bacterial host can affect plasmid stability, we measured the loss of
the IncP-1β plasmid pB10 in 20 different strains belonging to the α-,
β- and γ-Proteobacteria, including environmental isolates and lab
strains. Three strains showed a very rapid plasmid loss, four showed
slower plasmid loss, and for all other strains segregants where either
detected sporadically or not at all. This study shows that the same
plasmid can show high variability in stability in closely related
organisms, suggesting that strain specific host-plasmid interactions
can influence the persistence of a plasmid.
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Modeling the Spatial Dynamics of Plasmid
Transfer
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The long-term goal of this
study is to understand the population biology of transmissible plasmids in
spatially structured microbial communities: What are the mechanisms that
drive the horizontal transfer and persistence of these mobile genetic
elements? Why do they persist, even in the absence of selection for any of
the genes they carry? How does the spatial structure of natural microbial
communities influence the ecological and evolutionary dynamics of
plasmid-bacteria interactions? The current understanding of plasmid
population dynamics is almost entirely based on mathematical models that
simulate plasmid transfer and maintenance in completely mixed environments,
and there are no realistic models for spatially structured populations.
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.
Undergraduate and graduate students or postdoctoral researchers interested in working
on these or related projects can
email Dr. Top.
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