This project was prepared as part of a BioQUEST faculty development workshop entitled ASM/BioQUEST Bioinformatics Institute at American Society for Microbiology in March 2007. The BioQUEST Curriculum Consortium is committed to the reform of undergraduate biology instruction through an emphasis on engaging students in realistic scientific practices. This approach is sometimes characterized as an inquiry driven approach and is captured in BioQUEST's three P's (problem-posing, problem-solving, and peer-persuasion). As part of this workshop groups of faculty were encouraged to initiate innovative curricular projects. We are sharing these works in progress in the hope that they will stimulate further exploration, collaboration and development. Please see the following links for additional information:
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Attendees of the 2007 ASM/BioQUEST Bioinformatics Institute, American Society for Microbiology Headquarters, March 8 - 11, 2007
Hypotheses
1) arsC evolved after araA/araB since a reducing environment existed pre-dating the necessity for the reductase activity encoded by arsC.
2) ArsA, arsB and arsC should be highly conserved given their ubiquity in all three domains.
3) Non-ATP dependent AraB should be structurally different from ATP-dependent AraB.
4) The phlyogenetic relationships between ara genes should reflect the 16S rRNA phylogenies.
Results
1) arsC sequences are very dissimiliar between Archaea and bacteria. Indeed, sequences search do not recover any ArsC Archaea genes, although several species are known to contain them.
2) ArsA and arsB phylogenetically grouped with species in the Eubacteria, Archaea and Eukarya domains suggesting that other than structural constraints around the active site, there is much less conservation elsewhere.
3) The branches in the phylogenetic trees are very short indicating little conservation of sequence.
Student research
Bioinformatics
1)Do a thorough search for ArsC sequeneces. Most likely there will be new sequences online as interest in Archaean speicies is high.
2) Syntenic chromosome structure for the ars operon should be quite interesting since lateral gene exchange and homologous recombination have driven evolution of arsenate resistance across domains.
3) ArsA has an internal duplication of ATPase domains in most species investigated. It would be most interesting to explore this feature of gene structure.
Biochemistry
1) Mutations have been documented in E coli. Biochemical analysis of the relationship of arsenate resistance to gene structure will clarify structural constraints.
2) Selection of arsenate resistance mutants, using recombinant plasmids with heterologous sequences are all techniques that are accessible to student research.
NCBI Taxonomy Trees
NCBI Cn3D Protein Images
NCBI CDART Architecture Viewer
References
Crameri A, Dawes G, Rodriguez E Jr, Silver S, Stemmer WP. 1997. Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat Biotechnol.:15(5):436-8.
Colin R. Jackson and Sandra L. Dugas. 2003. Phylogenetic analysis of bacterial and archaeal arsC gene sequences
suggests an ancient, common origin for arsenate reductase. BMC Evolutionary Biology. 3:18-28.
Diorio, C., J. Cai, et al. 1995. "An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria." J. Bacteriol. 177: 2050-2056.
Marchler-Bauer A, Panchenko AR, Shoemaker BA, Thiessen PA, Geer LY, Bryant SH. 2002. CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 30(1):281-3.
Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, Ke Z, Krylov D, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Thanki N, Yamashita RA, Yin JJ, Zhang D, Bryant SH. 1997. CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res. 35(Database issue):D237-40. Epub 2006 Nov 29.
Saltikov, C. W. and B. H. Olson (2002). "Homology of Escherichia coli R773 arsA, arsB, and arsC Genes in Arsenic-Resistant Bacteria Isolated from Raw Sewage and Arsenic-Enriched Creek Waters." Appl. Environ. Microbiol. 68(1): 280-288.
Sato, T. and Y. Kobayashi. 1998. "The ars Operon in the skin Element of Bacillus subtilis Confers Resistance to Arsenate and Arsenite." J. Bacteriol. 180(7): 1655-1661.
Zhou, T., B. P. Rosen, et al. 1999. "Crystallization and preliminary X-ray analysis of the catalytic subunit of the ATP-dependent arsenite pump encoded by the Escherichia coli plasmid R773." Acta Cryst. D55: 921-924.
Future work
1) It is predicted that species that lack ArsA will have an ArsB gene encoding a non-ATPase dependent antiporter. This should be reflected in sequence analysis of the 2 different types of ArsB.
2) The lack of arsC genes in Archaea will be addressed by future genome sequencing projects.
3) ArsA and arsB have structural constraints (e.g. active site) so that these regions are conserved; however, structural constraints are relaxed.
4) The role of mutational analysis has not been addressed and should show insights into the structural constraints for arsC and arsB.