Dr. Valérie de Crécy-Lagard, Assistant Professor

Research topics for student projects: Identifying the function of every gene in all sequenced organisms is one of the major challenges created in the post-genomic era and is one of the steps that will enable successful systems biology approaches. This objective is far from accomplished. By different estimates, over 30-50% of the genes of any given organism are of unknown function, incorrectly annotated or given a broad nonspecific annotation. With more than 350 genomes sequenced and 1220 in the pipeline, unknown or incorrectly identified genes are propagating at an exponential rate making it all the more difficult to extract correct functional information. Without a specific functional annotation effort, the genome information generated to date might become even more difficult to analyze and greatly underexploited. Combining comparative genomics with experimental validation is a very efficient way to link gene and function. This combined approach has been the subject of our research program to discover new enzymes since the first genome was published. Several projects are ongoing in the laboratory centered on combining comparative genomics with genetic and biochemical studies to identify and functionally characterize many genes of unknown function involved in tRNA modification, coenzyme metabolism and other central metabolic pathways.

Research approaches and techniques: Methodological approaches include molecular manipulations aimed towards generation of mutants of Haloferax volcanii genes, site directed mutagenesis to identify key residues in novel enzymes, classical and molecular genetic methods to construct and combine mutants in different bacterial models and in yeast and general microbial physiology experiments.

Impact and benefit: Students will be exposed to genetic manipulation of bacteria, archaea and yeast as well as to various comparative genomic analysis methods.

 

 __________________________________________

Dr. Julie A. Maupin-Furlow, Associate Professor

Research topics for student projects: Recent advances in proteomics have provided glimpses into life at the level of whole cells. From this, it is evident that gene expression profiles often do not correlate with those of protein. Thus, it is important to understand not only transcriptional networking but also post-transcriptional control when investigating global regulatory responses. Regulatory proteases such as proteasomes, rhomboid and site-2-type proteases are key in the quality control and regulated turnover of proteins and, thus, are central players in modulating proteome composition. We have and are continuing to characterize the physiological roles and biochemistry of a number of these regulatory proteases in the halophilic archaeon Haloferax voclanii. Despite the fact that archaea are prokaryotes, much of their information machinery (DNA replication, transcription, translation) and protein quality control mechanisms (proteasomes, etc.) are more closely related to their eukaryotic than eubacterial counterparts. The archaeon H. volcanii serves as an ideal model system to investigate many of these key processes including regulated proteolysis, based on its complete genome and internationally established biochemical and genetic exchange tools. In our work, hypotheses concerning the molecular and biochemical functions of archaeal regulatory proteases will be tested using H. volcanii as a model organism. The proteases under investigation include: (a) proteasomes and proteasome-activating ATPases, (b) rhomboid proteases, and (c) site-2-type proteases. Students participating in the REU will purify and characterize the activity and partners of these proteases from recombinant H. volcanii using affinity tags. Other student projects will include the construction of site directed mutants of these proteases to probe structure-function. In addition, the biological roles of the proteases will be investigated through the construction of isogenic mutant strains and analysis of their phenotypes including identification of differentially produced proteins.

Research approaches and techniques: Methodological approaches include molecular biology and protein biochemistry to generate, purify and assay archaeal regulatory proteases and site-directed variants. In addition, targeted chromosomal gene knockout will be used to generate isogenic protease mutant strains of H. volcanii for phenotype analysis including growth and proteome composition, as assessed by 2Dgels coupled with mass spectrometry (e.g. MS/MS ESI-QSTAR).

Impact and benefit: Students will be exposed to the physiology of halophilic archaea, as well as to various methods in the biochemistry, genetics, molecular biology, and proteomics of this unusual and interesting group of microorganisms.

 

 __________________________________________

Dr. Wayne L. Nicholson, Associate Professor

Research topics for student projects: A major issue in Astrobiology is the search for signs and conditions of life on other bodies in our solar system such as Mars, Europa, or Enceladus. If life indeed exists on these bodies, it is likely microbial in nature. Recent research projects in our lab investigate the possibility of interplanetary transfer of microbes, their survival and proliferation in space and on other planets. Transfer between planets could be accomplished in two ways: (i) by natural asteroid or comet impacts which blast microbe-bearing rocks into space on planet-crossing trajectories; or (ii) by human space exploration activities which could carry Earth microbes to target planets or return extraterrestrial microbes to Earth. Bacterial spores of the species Bacillus are common inhabitants of rocks and common contaminants of spacecraft, and are able to survive the rigors of launch, spaceflight, and landing, therefore are considered ideal model systems for studying interplanetary transport. Specifically we have been testing the ability of spore-forming Bacillus spp. to survive interplanetary transfer conditions. In addition, we have been conducting experiments asking, if spore-forming bacteria are indeed deposited on Mars, can they survive, germinate, grow, and evolve in the Martian environment. These projects involve simulations of launch using low- and high-speed ballistics; exposure to space conditions using space simulators and actual space flights; atmospheric entry using sounding rocket flights; and exposure to Mars environmental simulations located at Kennedy Space Center. Our Astrobiology experimental program interfaces well with our overall fundamental goal of understanding the molecular bases of bacterial spore resistance properties.

Research approaches and techniques: Students participating in the REU will learn basic microbiological techniques; genetic approaches to the construction and analysis of Bacillus strains; operation of environmental simulators; spore survival assays; assays of individual and global phenotypic and transcriptional responses; and long-term evolution of bacteria in exotic environments.

Impact and benefit: Students will be exposed to a range of modern techniques in molecular microbiology and will be trained in the process of hypothesis-driven, experiment-based inquiry using the scientific method. In our experience we have noted that astromicrobiology research projects are particularly effective at exciting the imagination and enthusiasm of students. Over the years our projects have attracted and trained many top-notch undergraduate students leading to their authorship on peerreviewed publications (see recent Reference list for details) and further pursuit of rewarding careers in science and medicine.

Recent Peer-Reviewed Publications with Undergraduates:

Mastrapa, R.M.E., H. Glanzberg, J.N. Head, H. J. Melosh, and W.L. Nicholson. 2001. Survival of bacteria exposed to extreme acceleration: implications for panspermia. Earth and Planetary Science Letters 189: 1-8.

Nicholson, W.L. and B. Galeano. 2003. UV resistance of Bacillus anthracis spores

 

  __________________________________________

Dr. Jamie Foster, Assistant Professor

Research topics for student projects: Microbial mats are one of the oldest known community structures on Earth. These ancient communities are comprised of multi-species biofilms that form on various substrata in some of the most extreme environments on Earth. In my laboratory, students will be addressing three major questions associated with the formation and development of microbial mats found in the Highborne Cay, Bahamas (HBC). These major questions include: 1) what is the extent of microbial diversity associated with the HBC microbial mats and which species are essential for mat initiation and formation; 2) what are the underlying genetic and biochemical mechanisms required for mat formation to occur; and 3) determine the genetic and biochemical mechanisms by which these HBC mats can withstand long-term extended exposures to ultraviolet radiation (UVR).

Research approaches and techniques: Specifically, REU students will generate 16S rRNA gene clone and culture libraries to examine the full range of microbial diversity within the HBC microbial mats. Key mat forming species will be identified using in vitro modeling systems such as the flowcell in which artificial microbial mats can be formed and monitored in the laboratory. Nascent mats will be examined morphologically using various microscopy techniques (TEM, SEM, confocal, Laser Capture and light microscopy) as well as with molecular techniques (16SrRNA, FISH analysis). Those students that examine the expression of UVR stress response genes and their transcripts in the HBC mats will be trained in RTPCR, northern, and western blotting, HPLC, and GC/MS.

Impact and benefit: Students participating in this project will be exposed to the dynamic field of microbial ecology. Although fundamental laboratory skills are essential for a student research experience, it is also important that students grasp the larger concepts of microbial ecology and diversity. By working with HBC mat communities students will be exposed to an ecosystem in which hundreds of organisms interact and coordinate their activities to produce a complex biogeochemical cycling of their environment.

 

  __________________________________________

Dr. James F. Preston, Professor

Research topics for student projects: Research projects include: 1) Metabolic engineering of bacteria for utilization of lignocellulosics; selection of bacterial enzymes for the depolymerization of plant biomass and its fermentative conversion to alternative fuels and biobased products. Primary objectives are directed at the selection of genes that encode enzymes that process the glucuronoxylan polymers of the hemicellulose fraction of hardwoods and crop residues. Identification, cloning, sequencing of these genes will be followed by their expression in model bacteria, including strains of Escherichia coli and Bacillus subtilis, to engineer bacteria for efficient conversion of lignocellulosic resources to ethanol and lactic acid. Student projects may identify genes encoding endoxylanases from thermotolerant bacteria, clone and sequence these genes, and express them in a model bacterium. Additional projects may address the characterization of recombinant xylanases to determine substrate preferences, kinetic properties, and structural properties that define their application as catalysts for the depolymerization glucuronoxylans. 2) Definition of molecular mechanisms involved in the biocontrol of phytopathogenic nematodes by Pasteuria spp. The species Pasteuria penetrans is an endospore-forming bacterium that propagates as an obligate parasite of root-knot nematodes associated with several important agricultural crops, e.g. peanuts, tomtoes, peppers. Genome sequencing and bionfomatic tools are used to identify virulence factors required for their development as biological nematicides. Student projects may identify genes that encode adhesin proteins that are candidates for attachment of endospores to a particular Pasteuria species as required in initiating the infection of and propagation in the nematode host. Genes encoding adhesins will be cloned and overexpressed in model bacteria to produce quantities for biochemical characterization. These adhesins will be evaluated as encumbering agents that inhibit the ability of soil-borne nematodes to infect their host plants.

Research approaches and techniques: Research project 1) Approaches to identify thermotolerant endoxylanases will include PCR amplification of target genes, cloning target genes into plasmids for sequencing, and subcloning target genes into plasmids for high level expression in model bacteria. Approaches to characterize enzymes will utilize column chromatography to purify enzymes, spectrophotometry to assay enzymes, high-performance liquid chromatography to separate products for characterization, and NMR spectroscopy to chemically define products. Research project 2) Approaches to identify adhesin genes will utilize bioinformatic analysis with sequenced genomes from different Pasteuria spp., apply PCR amplification to identify these genes in genomic libraries, and subclone candidate genes for overexpression in model bacteria. Immunochemical techniques will be used to quantify production and attachment to nematode hosts.

Impact and benefit: Students will gain experience in experimental design, current techniques, and skill in data analysis and interpretation, and will present accomplishments through oral presentation and in a research paper formatted for publication in a research journal. The results of theses studies will contribute to the achievement of objectives of projects currently funded by either DOE or USDA.

 

 __________________________________________

Dr. Madeline Rasche, Associate Professor (2008 at NSF)

Research topics for student projects: The one-carbon metabolism of methaneproducing (methanogenic) archaea and methylotrophic bacteria contribute significantly to the cycling of carbon in environment, the biodegradation of wastes and toxic pollutants, and the production of methane as a greenhouse gas and energy source. To support these interesting metabolic pathways, methanogens and methylotrophic bacteria produce coenzymes such as methanopterin and methanofuran, which are active in pathways of one-carbon catabolism. Our laboratory focuses on understanding the genetics and biochemistry of coenzyme biosynthetic pathways in archaea and methylotrophic bacteria. One aspect of the project that is particularly well suited for summer research internships is the application of site-directed mutagenesis to investigate structure-function relationships in two methanopterin biosynthesis enzymes called RFAP synthase and dihydromethanopterin reductase. REU interns will use site-directed mutagenesis to alter individual amino acids suspected to play key roles in substrate specificity or catalysis. The altered proteins will be produced in Escherichia coli, purified, and biochemical characterized as described previously (reference). In addition, interested students will have the opportunity to further characterize enzyme using biophysical techniques.

Research approaches and techniques: Our research uses combined biochemical, genetics, and biophysical approaches to identify and characterize structure-function relationships of enzymes in the pathways of archaeal coenzyme biosynthesis. Students will gain experience in bioinformatics and comparative genomics, recombinant DNA technology, production of engineered proteins, protein purification, fast protein and high pressure liquid chromatography (FPLC and HPLC), enzymatic kinetics, site-directed mutagenesis, and selected biophysical techniques (microcalorimetry, electron paramagnetic resonance, and mass spectrometry) through collaborations with our colleagues in the UF Chemistry Department and at the UF Medical School.

Impact and benefit: Students will gain experience in laboratory and critical thinking skills that can provide a foundation for future research in microbiology, biochemistry, and genetics. The results obtained by the REU researchers will contribute to elucidating a fundamental biochemical pathway and may contribute to the broader goal of ehancing or modulating the process of biological methane production in the environment.

 

 __________________________________________

Dr. Nemat O. Keyhani, Associate Professor

Research topics for student projects: As critical constituents of fungal cell envelopes, hydrophobins are unique proteins that function in a diverse array of physiological processes. Amongst the most surface active biomolecules described to date, one of their functions is to decrease the surface tension of water allowing fungi to grow into the air. Their remarkable biophysical properties are also of great interest for use in (nano) biotechnology and various biomedical applications. We have isolated and expressed in E. coli three hydrophobins from the entomopathogenic fungus, Beauveria bassiana, implicated in a diverse range of physiological functions. Hypotheses concerning the molecular and biochemical functions of hydrophobins will be tested. Related research projects involve interactions, including the roles of the conidial envelope (hydrophobin layer), between fungi and host cells. The range of fungi under investigation include (a) entomopathogens such as B. bassiana and Metarhizium anisopliae and their interactions with a variety of insect cell lines, and (b) the human opportunistic pathogen Aspergillus fumigatus and their interaction with macrophages. Students participating in the REU will construct site directed mutants of the hydrophobins in order to probe structure-function issues. Other student projects involve determining cytotoxic effects (by microscopy and other cell biological methods) of various fungi on host (tissue culture) cells lines.

Research approaches and techniques: Methodological approaches include molecular manipulations aimed towards generation of mutants, and protein expression and purification aimed towards biophysical and structural characterization of the hydrophobin proteins, and cell biology/microscopy to examine the interactions between fungi and (insect and mammalian) host cells.

Impact and benefit: Students will be exposed to biochemical and genetic manipulation of fungi, as well as to various methods in cell biology and host-parasite interaction.

 

 __________________________________________

Dr. Zhonglin Mou, Assistant Professor

Research topics for student projects: The PP pathway not only produces three metabolites, ribose-5-phosphate, erythrose-4-phospahte and sedoheptulose-7-phosphate, but also produces the reducing equivalent NADPH. NADPH produced by the PP pathway is essential for maintaining the redox potential to protect against oxidative stress. Many human diseases are directly associated with compromised NADPH production through the PP pathway. For example, deficiency in G6PDH, which catalyzes the first committed step of the pathway,causes a spectrum of diseases including neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. In plant cells, the PP pathway activity is increased upon pathogen infection, and the activity is required for salicylic acid (SA)-induced redox changes, NPR1 protein reduction, and PR gene expression. We are interested to study the regulation of plant defense responses by the PP pathway. We have identified T-DNA insertion mutants for most of the PP pathway genes, and have performed preliminary characterization of defense-related phenotype of the mutant plants. Students participating in the REU will make plant expression constructs to overexpress the PP pathway genes in Arabidopsis. Other student projects involve plating Arabidopsis seeds on MS plates, infecting the Arabidopsis plants with bacterial pathogens, and monitoring the growth of the pathogens inside the plants.

Research approaches and techniques: Methodological approaches include molecular manipulations aimed towards generation of transgenic plants, and Arabidopsis mutant isolation and characterization, and cell biology/microscopy to examine the interactions between bacterial pathogens and plant cells.

Impact and benefit: Students will be exposed to genetic and biochemical manipulation of plants, as well as to various methods in cell biology and plant-microbe interaction.

 

 __________________________________________

Dr. Eric W. Triplett, Professor

Research topics for student projects: Undergraduates will work on our NSF-funded project to better understand the association between plants and nitrogen-fixing bacterial endophytes. This work is based on our discovery of nitrogen fixation in wheat provided by Klebsiella pneumoniae 342 (Iniguez et al. 2004). We are determining the mode of invasion of this bacterial endophyte into the host plant, the timing of the onset of nitrogen fixation in the plant, and the location of bacterial nitrogenase expression within plant tissues. Students will also work with our graduate students in our signature tagged mutagenesis (STM) of Kp342 to identify genes needed for endophytic colonization. In addition, undergraduates will assist us with experiments to determine the importance of the nitrogen-fixing phenotype on plant colonization and on the ability of Kp342 to compete with other endophytic bacteria.

Research approaches and techniques: Methodological approaches include scanning laser confocal microscopy, in situ hybridization, mutant construction, and mutant screening. Some bacterial identification will also be done using 16S rRNA sequencing and physiological analyses. The students will also learn to use our genome database in the analysis of the STM mutants.

Impact and benefit: Students will be exposed to physiological and genetic analysis of bacteria. They will also learn how to use genome data to ask specific questions regarding host-microbe interactions.

References: Iniguez, A.L., Y. Dong, and E.W. Triplett. 2004. Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Molec. Plant-Microbe Interact. 17:1078-1085.

 

__________________________________________

 Dr. Lonnie Ingram, Distinguished Professor

General area: Global redirection of central metabolism by genetic engineering;  Industrial fermentation processes;  Carbohydrate metabolism;  Expression and secretion of glycohydrolases which degrade plant polymers;  Alcohol tolerance.

Research topics for student projects:  Our work focuses on the genetic engineering of novel bacterial biocatalyst for the conversion of lignocellulosic biomass into ethanol fuels and other fermentation products which can replace imported petroleum. This work involves cloning and moving genes between bacteria to add new and useful traits to ethanol producing organisms, design of novel engineering processes for the production of ethanol (and other chemicals), nutritional investigations, and the identification of genes which contribute to ethanol tolerance.  Global gene analysis is being used to investigate fundamental processes in metabolically engineered bacteria.

Research approaches and techniques: 

Impact and benefit: 

 

 __________________________________________

Dr. Joseph Larkin III, Assistant Professor

Research topics for Student Projects:  The basic goal of the mammalian immune system is the elimination of harmful, infectious microorganisms.  Hallmarks of an effective immune response include: the ability to devise and mount a timely defense strategy that is specific to diverse infectious microorganisms, the ability to stop the attack once the danger subsides, and the ability to limit damage to self tissues.  Our laboratory focus is directed towards gaining a better understanding of mechanisms by which the immune system minimizes damages to self-tissues, a process called tolerance.  In general, immune system tolerance is highly effective; however the self-tissue damage that occurs in rheumatoid arthritis, type I diabetes, multiple sclerosis, and lupus is mediated by aberrant immune responses. Recently, a subset of immune system cells, known as regulatory T cells, have been shown to play a significant role in moderating immune responses.  However, it is not clearly understood how variations in the environment where regulatory T cells develop, absolute regulatory T cells numbers, the activation environments of regulatory T cells, and the ability of regulatory of regulatory to migrate within the body influence regulatory T cell functions. Student projects will include: 1) the characterization of the regulatory T cell population in a mouse model where immune system damage to self-tissues result in death, 2) examination of regulatory T cell function under a number of activating conditions, and 3) examination of intracellular processes occurring within regulatory T cells during effective immune regulation.

Research approaches and techniques:  Our laboratory is currently examining regulatory T cell function on the intracellular, cellular, and the preclinical (mouse model) levels.  Students will gain a global perspective of immunological research in the area of immune system tolerance.  Students will contribute to ongoing studies in the lab and will participate in in vivo mouse experiments, flow cytometry, cell culture, proliferation assays, ELISA, electrophoresis, western blotting, and PCR.  The undergraduates that work on these projects may become co-authors of publications resulting from these studies.

Impact and benefit:  Students will be given opportunities to directly contribute in translational research of potential benefit to humanity.  Students will be encouraged to think critically and independently with regards to their research project.  Students will develop communication skills while participating in weekly laboratory meetings and journal clubs.

 

_______________________________________

Dr. Claudio F Gonzalez, Assistant Professor

Research topics for student projects: My group is interested in the characterization and the identification of the biological importance of enzymes without specific annotations and not assigned to any particular biological role. Our current projects are related with the study of a family of enzymes with close related enzymatic properties; the esterases. This big family includes carboxylesterases, aryl esterases and thioesterases. Elucidate the biological role of unknown enzymes with esterase activity represents a big challenge due its broad substrate specificity. Because substrate-enzyme interactions are quiet difficult to predict we utilize a subset of functional methods to get important biochemical clues to pursue our goals. Using a combination of panels of esters with different chemical scaffolds we can understand the substrates preferences of these enzymes. Estereases are involved in different cellular processes from biosynthetic pathways to xenobiotics degradation. Several of them are useful in several industrial applications like stereochemistry or degradation of recalcitrant environmental contaminants. Students will participate in one of the following ongoing projects in our lab: purification of predicted esterases, characterization of the enzymatic profile of feruloyl ester hydrolases or selected Escherichia coli carboxylesterases.

 

Research approach and techniques: The methodology to be used include, cloning, overproduction and purification of the enzymes of our interest. Students will use different biochemical methods to determine the biochemical parameters of purified esterases. The impact of the deletion of the genes encoding these proteins will be evaluated in model bacteria, like Escherichia coli and Pseudomonas aeruginosa.

 

Impact and benefit: The students will be involved in the utilization of several modern techniques in genetic manipulation, protein purification and biochemical characterization. As part of the laboratory group they will participate in our weekly scientific meetings.