Research Projects in the Liles Laboratory

Our research group works at the intersection of environmental and clinical microbiology. We are using a combination of modern molecular biological tools and classic microbiological techniques to 1) Discover novel antibiotic compounds and antibiotic resistance mechanisms produced by environmental microorganisms, 2) Identify and characterize biological control agents to control disease in channel catfish, and 3) Study the complex microbial communities in soils, activated sludge, bioreactors and freshwater streams and their response to environmental factors.

Bacterial and Viral Community Genomics

The history of microbiology as a science has traditionally relied upon microscopy and the ability to culture microorganisms on a petri dish.  The advent of molecular microbiology has brought new tools into the repertoire of microbiologists--now we are armed with whole genome sequences of the bacteria that we study, and can use a combination of microscopy with molecular probes.  Rather than supplanting the techniques that have been used for over 150 years of microbiology, the molecular era has given new life and new approaches to the field.

The work of Carl Woese and colleagues demonstrated that the 16S ribosomal RNA gene is conserved among all life forms.  Molecular phylogenetic analysis now allows an objective and quantitative assessment of evolutionary relationships, and has revealed the vastness of microbial diversity on planet Earth.  Despite only being able to study a small percentage of the microbial life on Earth, cultured microorganisms have provided us with many useful natural products, such as antibiotics. Yet even to this day there remains an overwhelming diversity of microbial life that is unknown to science. Perhaps the greatest challenge for microbiologists in the 21st century is to learn more about this "silent majority" of microbial life alive on our planet. 

Show below is a figure from an excellent review that demonstrates the large gaps in our current knowledge, as each of the bacterial divisions shown without shading do not have a single cultured representative:

If the phylogenetic diversity of microbial life is so vast, how much unexplored metabolic and functional diversity among microbial communities is left to discover?  To tap into this genetic potential our lab has collaborated with the labs of Dr. Bob Goodman at Rutgers University and Dr. Jo Handelsman at the University of Wisconsin-Madison. We use an approach that is designated ‘metagenomics’, which is the analysis of the collective genomes of the microorganisms in a natural environment. By cloning DNA isolated from diverse bacteria or viruses directly into a culturable host (e.g., E. coli) the genetic diversity of microorganisms in natural environments may be accessed and natural products produced by these environmental microorganisms may be expressed within a surrogate E. coli host.  The metagenomic libraries can be screened by direct sequencing, identifying recombinant clones that contain a specific gene sequence, or by functional assays. All three of these screening methods are used in the Liles laboratory to study bacterial and viral communities in soils, activated sludge, and bioreactors. 

Although metagenomic analysis as a field is still in its infancy, a number of exciting discoveries have been made that indicate that a wealth of chemistry and biology is waiting to be unearthed. We constructed a number of libraries that collectively contain many Gigabases of 'environmental' DNA from soils, activated sludge, and bioreactor communities.  We have found two structurally related, novel antibiotics, designated turbomycin A and turbomycin B, with broad spectrum activities.  and numerous 16S rRNA genes that indicate that a vast diversity of organisms, including many that diverge deeply from cultured microbes, contributed DNA to the library. The 16S rRNA genes or other phylogenetic indicators, sequence analysis, and functional screening provide the opportunity to link phylogeny and function. Ultimately, this information may provide the basis for strategies to culture the organisms from which the DNA was derived or may provide the basis for in situ probing to determine when, where, and with whom the genes are expressed.

Three projects in the Liles laboratory rely upon a community genomic approach:

A.  Antimicrobial discovery project

In collaboration with the Lucigen Corporation (Madison, WI) the Liles laboratory is developing new metagenomic libraries and screening libraries for the presence of antibiotic compounds with activity against bacteria classified as potential biological weapon agents by the National Institutes of Health and the Center for Disease Control.

Please contact Dr. Liles for further information.

B. Horizontal transfer of antibiotic resistance in activated sludge and bioreactor communities

In collaboration with Dr. Willie F. Harper, Jr. (Dep. Environmental Engineering, University of Pittsburgh) our lab is investigating mechanisms by which bacteria within these environments can confer antibiotic resistance to other bacteria species (i.e., horizontal gene transfer).  This culture-dependent and -independent approach is evaluating plasmid-mediated antibiotic resistance, bacteriophage transduction of resistance, and community genomic approaches to evaluate chromosomally-encoded resistance to a diverse panel of antibiotic compounds.

C.  Viral community genomics in soils and activated sludge

As with the bacterial life in soils, the population of bacterial-specific viruses, or bacteriophage, that infect these soil bacteria are phenomenally diverse. Bacteriophage have genomes considerably smaller than that of a bacteria; most are less than 100kb, and express the factors necessary for infection, replication, and lysis of the bacterial host cell. Many bacteriophage express lysins that will literally "burst open" an infected bacterial cell, and thus are useful in controlling certain pathogenic bacteria.

Our initial projects involve isolating diverse bacteriophage from soils and directly isolating their nucleic acids (i.e., both DNA and RNA viruses). In collaboration with the Lucigen Corporation (Middleton, WI) we have constructed several soil and activated sludge bacteriophage community genomic libraries.  Sequence analysis of these viral community genomic libraries reveals great genetic diversity within the viral communities, and less than half of the sequences hit significant homologs in the GenBank nr/nt database.  Linking culture-dependent and culture-independent approaches to describe viral communities is an ongoing line of investigation, as well as characterizing phage-derived clones that confer antibiotic resistance.

Biological control of disease in channel catfish

Bacterial pathogens cause millions of dollars in losses annually for the aquaculture industry in the Southeastern United States.  In collaboration with the laboratories of Dr. Jeff Terhune (AU Dept. Fisheries and Allied Aquacultures), Dr. Joseph Kloepper (AU Dept. of Entomology and Plant Pathology) and Dr. Joseph Newton (AU Dept. of Pathobiology), we have identified bacteria and bacteriophages that are antagonistic to the bacterial pathogen Edwardsiella ictaluri.  Current studies are aimed at evaluating and enhancing the protective effects of biological control agents in preventing mortality in aquaria-raised channel catfish and aquaculture ponds.

Bacterial diversity and population structure in natural enviroments

Identifying microorganisms present in a natural sample is important for researchers in food safety, environmental monitoring, infectious disease, and ecological studies, among other disciplines. There are many different techniques available for determining the diversity of bacterial taxa in an environmental sample (e.g., T-RFLP, ARISA, DGGE, etc.), which typically rely upon PCR amplification of the 16S rRNA gene and distinguishing between the various ribotypes present within the sample.  Each technique has its own advantages and disadvantages, all allow a fairly rapid determination of changes in bacterial community structure, without the more laborious efforts involved in metagenomic library construction and analysis.

A.  Phylosphere Microbial Communities in Freshwater Streams

In collaboration with Dr. Jack Feminella (AU Dep. Biological Sciences) we are examining the succession of bacterial and fungal communities on leaves submerged in freshwater streams, examining the effects of time of incubation, access to macroinvertebrates, and other environmental variables on phylosphere communities.  Multiple experimental approaches are being taken, linking knowledge of microbial communities with freshwater ecology.

B.  Phylogenetic Microarrays "Phylochips" to Identify Changes in Soil Bacterial Populations

Using custom microarrays synthesized by the Nimblegen Corporation (Madison, WI; http://www.nimblegen.com/) we have targeted the 16S rRNA gene of diverse soil bacteria in order to simultaneously detect the presence of multiple bacteria in a natural sample. Pictured is an actual example of the results of an array hybridization, demonstrating the large dataset of information derived from microarrays.  In collaboration with Dr. Nedret Billor (AU Dep. of Mathematics and Statistics), the data from Phylochip hybridizations have been analyzed to address how in silico predictions of probe hybridization kinetics effect our data, and in the phylogenetic resolution achieved by this method.

This image represents approximately 1/16th of a microarray's total area