Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Shewanella Federation
41
The Microbial Proteome Project: A Database of Microbial Protein Expression in the Context of Genome Analysis
Carol S. Giometti1* (csgiometti@anl.gov), Gyorgy Babnigg1, Sandra L. Tollaksen1, Tripti Khare1, Angela Ahrendt1, Wenhong Zhu2, Derek R. Lovley3, James K. Fredrickson4, and John R. Yates III2
1Argonne National Laboratory, Argonne, IL; 2Scripps Research Institute, La Jolla, CA; 3University of Massachusetts, Amherst, MA; and 4Pacific Northwest National Laboratory, Richland, WA
This project, funded through the Microbial Genome and Genomics:GTL programs, is focused on the detection and characterization of differential protein expression in microbial systems relevant to the goals of the Office of Biological and Environmental Research. As part of this effort, relational databases are being used to assimilate and integrate the data collected. This growing knowledgebase can serve as a resource for comparison of proteomes across species and assessment of differential cellular responses to a variety of growth conditions. The Microbial Proteome Project knowledgebase currently includes experimental details and proteome data for 11 different microbes (Deinococcus radiodurans, Geobacter sulfurreducens, Geobacter metallireducens, Methanococcus jannaschii, Prochlorococcus marinus, Pyrococcus furiosus, Psychrobacter sp.5, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Shewanella oneidensis, and Synechocystis sp. PC) and includes over 8000 protein pattern images that are accessible to authenticated users.
As partners in the Shewanella Federation and University of Massachusetts Genomics:GTL projects, our laboratory efforts in the past year have been focused on the proteomes of S. oneidensis MR-1, Geobacter sulfurreducens, and Geobacter metallireducens. Using two-dimensional electrophoresis (2DE) analyses, we have been able to complement the results generated using LC/MS-MS proteomics and microarray mRNA analysis methods, providing “snapshots” of protein components to confirm the expression of specific proteins, measure their relative abundance, and detect post-translational modifications. In addition to traditional 2DE methods, we have introduced approaches that capitalize on the sensitivity of fluorescence and chemiluminescence. Using these approaches, we have been able to detect differences in protein phosphorylation patterns and in heme protein expression. By combining affinity chromatography to enrich samples for phosphoproteins with detection using phosphate-specific fluorescent dyes and immunoblotting with antibodies against specific phosphoamino acids, we have detected differences in the phosphoproteome of S. oneidensis when cells are grown aerobically compared to anaerobically. To optimize our detection of c-type cytochromes, we have used an isoelectric focusing fractionation protocol to concentrate G. sulfurreducens and G. metallireducens protein samples prior to electrophoresis and then detected the heme-positive proteins using a chemiluminescence assay. This latter method has increased the number of heme-positive proteins detectable from only four or five up to approximately 25 for each Geobacter species, and has provided the opportunity to do comparative analysis of the c-type cytochromes expressed by each of these microbes grown under different conditions.
In parallel with our investigation of subsets of the microbial proteomes, we continued our efforts to identify both constitutively expressed and induced proteins in S. oneidensis, G. sulfurreducens, and G. metallireducens. Our knowledgebase currently includes the identifications of 575 S. oneidensis MR-1, 400 G. sulfurreducens PCA, and 77 G. metallireducens proteins together with the peptide mass data used to obtain these identifications. These identifications represent the most abundant protein spots detected in 2DE patterns with either Coomassie Blue R250 or silver nitrate and include proteins expressed in S. oneidensis cells grown with 50% dissolved oxygen (i.e., aerobic conditions), 0% dissolved oxygen (i.e., suboxic or hypoxic), Fe(III), nitrate, or fumarate, G. sulfurreducens cells grown with either fumarate or Fe(III), and G. metallireducens cells grown with either nitrate or Fe(III). When the functional categories of the identified S. oneidensis and G. sulfurreducens proteins are compared, the relative number of identified proteins in each category is quite similar. Exceptions are found in the categories of cellular processes and protein fate with a greater percentage of S. oneidensis proteins identified than G. sulfurreducens proteins. More proteins associated with regulatory functions, on the other hand, have been identified among the G. sulfurreducens proteins detected in 2DE patterns than among the S. oneidensis proteins. Also, a larger number of G. sulfurreducens proteins with no annotation or annotated as having unknown function have been identified whereas more proteins annotated as hypothetical proteins has been identified in the 2DE patterns of S. oneidensis. The latter observation could reflect the less mature annotation of the G. sulfurreducens genome compared to the S. oneidensis genome, i.e., proteins currently having no functional annotation could be reannotated as proteins with known function as improved methods for functional annotations become available. Of the 77 G. metallireducens proteins identified, 25 are currently annotated as unknown, hypothetical, or conserved hypothetical proteins.
Since the overall goal of this project is to provide a public resource of protein expression information for microbes in the context of genome sequence, in addition to a secure website shared by Shewanella and Geobacter co-investigators, public websites have been designed to provide access to proteome analysis results as they become validated and published. ProteomeWeb (http://ProteomeWeb.anl.gov) is an interactive public site that currently provides the identification of M. jannaschii proteins detected by 2DE with links to genome sequence information, tools for mining the proteome data, and links to metabolic pathways. GelBank (http://GelBank.anl.gov) includes the complete genome sequences of approximately 205 microbes and is designed to allow queries of proteome information. Numerous tools are provided, including the capability to search available sequence databases for specific protein functions and amino acid sequences. Web applications pertinent to 2DE analysis are provided on this site (e.g., titration curves for collections of proteins, 2DE pattern animations). The database is currently populated with protein identifications from the Argonne Microbial Proteomics studies and will accept data input from outside users interested in sharing and comparing results from proteome experiments.
This work is supported under Contract No. W-31-109-ENG-38 with the U.S. Department of Energy.
* Presenting author
