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Functional genomics

Microbial genome sequencing

The availability of complete genome sequences from microorganisms, plants, animals and humans has marked a new age of biology, and it may lead to a revolution in various scientific fields, so our first research priority is to focus on sequencing and functional analysis of the microorganisms important to environmental cleanup, bioenergy, and  especially anaerobic microorganisms.

Because of their unique characteristics, the psychrophilic metal-reducing bacteria isolated by this group, Shewanella sp. PV-4, and Shewanella sp. W3-18-1, were selected for sequencing along with other 12 Shewanella strains through Shewanella Federation. The alkaliphilic isolate (Alkaliphilus metalliredigens) is also being sequenced by JGI.  The genome sequences from these isolates will provide information on the genetic differences that account for ecological success in different environments and support different physiologies, help to gain insight into the mechanisms and dynamics of evolution and speciation for metal-reducing bacteria, and determine whether core sets of genes exist for metal-reducing species. Understanding these key issues will greatly contribute to the successful, in-situ application of metal-reducing bacteria for bioremediation purposes, and advance our basic understanding of prokaryotic evolution and ecology using metal-reducing bacteria as a leading model. 

Due to their uniqueness, one of these 5 ancient Thermoanaerobacter strains (T. ethanolicus X514) is being sequenced by DOE Joint Genome Institute (JGI). For comparative analysis, a modern strain (T. ethanolicus 39E) is also being sequenced. By comparing the genome sequences, we should obtain insightful information on adaptive responses of microorganisms to the extreme environments with high temperature, high radiation, high pressure and low nutrients. The whole-genome sequences of thermophilic, iron-reducing bacteria will also shed lights on metal reduction in gram-positive bacteria. 

Clostridium cellulolyticum ATCC 35319 carries out a variety of processes including the biodegradation of cellulosic materials coupled to the production of fuels and chemicals, and are important in carbon cycling by contributing to the carbon flux at both local and global scales in natural environments. Thus we are also sequencing the genome of C. cellulolyticum to gain a better understanding of the mechanisms and regulations of microbial cellulose fermentation and address the goal of developing strategies to optimize metabolic conditions for cellulose conversion into energy and commodity products. 

Microarrays-based genome scale analysis

Large-scale sequencing of entire genomes represents a new age in biology, but the greatest challenge is to define gene functions and their regulatory networks at the whole-genome/proteome level. Under the support of various U.S. Department of Energy (DOE) programs, my laboratory is using both integrated genomic and proteomic approaches to study the functions and regulations of a range of environmentally important microorganisms. We have constructed whole genome microarrays for the following organisms: Shewanella oneidensis MR-1 (metal reduction), Deinococcus radiodurans R1 (radiation resistance), Rhodopseudomonas palustris (photosynthesis), Nitrosomonas europaea (ammonium oxidation), Desulfovibrio vulgaris (sulfate reduction), Geobacter metallireducens (metal reduction), and Methanococcus maripaludis (methane production). In collaboration with Dr. James Tiedje¡¯s lab, Exiguobacterium sp. 255-15 and Desulfitobacterium hafniense DCB-2 have also been constructed.  We have used them to systematically understand the functions and gene regulatory networks of these environmentally important microorganisms by collaborating with scientists from various universities, national laboratories and companies within the U.S. and from other countries.

Currently, the functional genomics studies in my laboratory are centered on the metal-reducing bacterium, S. oneidensis MR-1 as well as other Shewanella species, which is considered an E. coli-like model organism by DOE, and the sulfate-reducing bacterium, Desulfovibrio vulgaris. We have used microarrays to analyze gene expression under different growth and stress conditions, such as oxygen, nitrate, nitrite, salt, and temperatures. The microarray-based studies are a part of integrated studies along with proteomics and metabolomics. In addition, we are using microarrays to visualize genome differences among different sulfate reducing bacteria.

Now, microarray-based hybridization is a standard tool for monitoring gene expression, but it was difficult to analyze gene expression in prokaryotes several years ago. My laboratory was among the pioneering laboratories to demonstrate and validate the usefulness of microarrays for monitoring gene expression in prokaryotes. We have used the whole genome microarrays to identify genes expressed under a variety of growth and stress conditions, namely, heat stress, cold stress, high salt, low/high pH, oxidative stress, and metal toxicity, and to characterize genetic mutants involved in energy metabolism, global regulation and stresses. We also developed and used microarray hybridization to reveal genetic differences between closely related strains of microbes without sequencing.

Microarray-based genomic technology is very powerful for defining gene functions and regulatory networks as illustrated by one of our studies in Deinococcus. We used whole-genome microarrays to investigate the transcriptome dynamics of D. radiodurans recovering from ionizing radiation. Strikingly, a novel ATP-dependent DNA ligase involved in radiation damage repair was identified. A complex regulatory network involved in DNA repair was also revealed. Our results suggest that Deinococcus cells efficiently recover from radiation damages through complex coordinated regulatory network processes, including DNA repair, free radical minimization, switching energy metabolic pathways and biosynthetic pathways, and induction of cellular transport and cleaning systems. Such findings are not only important to understand DNA repair in this organism, but also important to medical studies. This work will be a pace-setting contribution to microbial functional genomics of radiation resistance.  

Mutagenesis

One of the most powerful ways to define the function of a gene is to turn the gene off or change the expression by replacing the normal gene with a mutated counterpart. We have successfully developed and utilized vector systems for homologous recombination in S. oneidensis MR-1. Currently, this laboratory is interested in understanding transcriptional gene regulatory network in S. oneidensis.  We are targeting approximately 220 annotated transcriptional factors (TFs) for knockout mutagenesis.  We have also systematically knocked out some of the genes involved in metal reduction as revealed by microarray analysis.

More than 40 deletion mutants have been generated using a PCR-based, in-frame deletion mutagenesis strategy, including some very important genes involved in global regulation such as etrA, arcA, fur, crp, fur/etrA, etrA/crp. Microarray-based gene expression profiling has been used to analyze a number of these mutant strains. For example, we have employed whole-genome DNA microarrays, liquid chromatography-mass spectrometry (LC-MS)-based proteomic method, and computational motif discovery tools to define the S. oneidensis Fur regulon. In contrast to the finding (Fur is local repressor) in E. coli, genomic studies suggest that Fur serves as a global regulator and as both repressor and activator in Shewanella.  In addition, as a part of the Shewanella Federation¡¯s efforts, we are generating deletion mutants for all 43 cytochrome genes annotated in S. oneidensis MR-1. 

Proteome profiling and cloning

By cooperating with scientists at Pacific Northwest National Laboratory, Argonne National Laboratory and Oak Ridge National Laboratory, we have also used 2-D gel and mass spectrometry to monitor protein expression profiles under different growth conditions with both wild type and mutant cells of S. oneidensis MR-1. Our results indicate that such analysis is very useful to reveal additional insights which microarray-based analysis could not provide.

Recent progress in whole-genome sequencing has provided information on the composition of many proteins from a variety of organisms, but the challenge remains to understand how proteins confer on cells their capabilities, structure, and higher-order properties. Along with mass spectrometry, phage display and yeast two hybrid systems are powerful techniques for studying protein-ligand interactions.  Thus we are developing capabilities for studying protein-ligand interactions in S. oneidensis MR-1 with emphasis on phage display and two hybrid systems.

The first key step of developing phage display and two hybrid systems is to clone all protein-coding ORFs into universal vectors, which is a very time-consuming and tedious process.  We have spent tremendous efforts in gene cloning.  So far, more than 4,000 genes have been cloned.

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