Introduction

1. The Meiothermus ruber (Thermales, Thermaceae) Genome Annotation Project - an authentic research experience for undergraduates in microbial genome analysis Scott, Lori R.1, Ghrist, Angela C. 2, Westemeyer, Blaine1, Petersen, Max1, Edison, Kristina1, Sieg, Alex1, Baumgartner, Angela1, Curtis, Troy1, Geison, Elizabeth1, Lehpamer, Nicole1, Oldfather, Nicole2, Allibone, Kevin1 and Sollenberger, Ryan1 1Augustana College, Rock Island, IL. 2Scott Community College/EICCD, Bettendorf, IA Conclusions Results Introduction Fig.2. The C-SCHR domain of this chromate transporter contains 5 transmembrane helices. This profile is the product of an amino acid sequence analysis using the TMHMM Server 2.06. The “Adopt-a-Genome” Education Program sponsored by the DOE Joint Genome Institute makes available to colleges/universities microbial genome sequence data for use in authentic research in genome annotation. Genome annotation identifies and attaches biological information to putative genes using bioinformatics technology. The microbes in the Adopt-a-Genome program are unusual and from sparsely investigated parts of the tree of life, so the likelihood of exciting discoveries and variations on the classical pathways is high. The long-term goal of the JGI’s Education program is to build on the annotation with bacterial characterization and functional genomics (e.g., insertional mutagenesis, protein overexpression with subsequent biochemical and biophysical characterization).1 The adopted organism Meiothermus ruber is an aerobic, Gram-, nonmotile, red-pigmented thermophile of the phylum Deinococcus-Thermus. In natural environments, Meiothermus strains are found in thermal limnetic systems, primarily in terrestrial hotsprings.2  The M. ruber genome was sequenced through a collaboration between the JGI and DSMZ.3 The Meiothermus ruber Genome Annotation Project is a network of regional 2-year and 4-year colleges/universities that are collaborating to annotate the ~3000 putative coding regions identified in the initial automated gene-calling analysis of the Meiothermus ruber genome. In this project, 11 students from two of the collaborating institutions contributed to this inaugural research experience, which included both computer-based annotation and benchtop components. The following questions were asked: Is there evidence to support the original functional prediction(s) of select M. ruber genes? In addition, could evidence of horizontal gene transfer and/or paralogs be identified? Could high quality genomic DNA be isolated from M. ruber that was suitable for PCR? Could select M. ruber genes be cloned into the pUC18 plasmid vector and transformed into E. coli for future functional genomics studies? Fig. 6. The Inquiry Wheel, a contemporary view of the scientific process.8 Questioning is placed as the center. Table 1: Bioinformatics analysis supported the original gene-call for 22 of the 23 ORFs studied within the M. ruber genome The DOE JGI’s Adopt-a-Genome Program provides undergraduates with an inexpensive and readily accessible authentic research experience in microbial genome annotation. By participating in the Meiothermus ruber Genome Annotation Project, students employed the scientific process as depicted in the Inquiry Wheel (Fig. 6). They entered the Inquiry Wheel with the question “Does the bioinformatics evidence support the original functional prediction(s) of their assigned genes?” and then traversed all stages of the wheel over the course of the project. Students confirmed the automated gene-call for 22 of 23 open reading frames, which included reclassifying one ORF as an unknown protein; 9 of 23 genes were identified as having paralogs; and horizontal gene transfer was proposed for a chromate transporter gene and a GCN5-related N-acetyltransferase gene (data not shown). Two M. ruber genes were confirmed as cloned into the pUC18 (chromate transporter and N-carbamoylputrescineamidase) by restriction enzyme analysis ,and by PCR and sequencing (data not shown). With ~3000 proposed genes in the M. ruber genome yet to annotate, there are many inquiry-based projects for future participants. In addition, functional genomics studies are needed to provide wet-lab confirmation of the proposed annotations. Firmicutes Proteobacteria Deinococcus-Thermus Cyanobacteria * Fig. 3 Incongruent phylogenetic tree and strong alignment hit to a distantly related species suggest horizontal gene transfer for the N-SCHR chromate transporter gene (coordinates 44519-45106). Organisms from the represented phyla are color-coded. This tree was constructed using Phylogeny.fr,7 which runs and connects various bioinformatics programs to reconstruct a robust phylogenetic tree from a set of sequences. T-Coffee program performed the final multiple sequence alignment. Literature cited Materials and methods 1DOE Joint Genome Institute’s Adopt-a-Genome for Education. Accessed 2010 April 2. http://www.jgi.doe.gov/education/genomeannotation.html 2Da Costa, M, Rainey, F & Nobre, M. 2006. The genus Thermus and relatives. In The Prokaryotes, 3rd ed. Vol. 7, pp.797-812. Edited by M. Dworkin, S. Falkow, E. Rosenberg, H. Schleifer & E. Stackebrandt. New York: Springer. 3DOE Joint Genome Institute’s Adopt-a-Genome Project, genome listings. Accessed 2010 April 2. http://www.jgi.doe.gov/education/adoptagenome/index.html 4 Integrated Microbial Genomes-Annotation Collaboration Tool (IMG-ACT/edu). Accessed 2010 April 2. http://img-act.jgi-psf.org/user/login 5Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 6Phylogeny.fr.: robust phylogenetic analysis for the non-specialist. 2003. Accessed 2010 April 2. http://www.phylogeny.fr/ 7TMHMM Server 2.0. Accessed 2010 March 15. http://www.cbs.dtu.dk/services/TMHMM/ 9R. Reiff, W. S. Harwood, T. Phillipson. "A scientific method based upon research scientists’ conceptions of scientific inquiry." Proceedings of the 2002 Annual International Conference of the Association for the Education of Teachers in Science, eds. Peter A. Rubba, James A. Rye, Warren J. Di Biase, Barbara A. Crawford. ERIC Document Reproduction Service No. ED (465 602). Question 1. Twenty-three M. ruber genes were annotated using the online IMG-ACT/edu bioinformatics toolbox created by the DOE JGI.4 The annotation process is organized by module and uses the following bioinformatics tools: Question 2. Genomic DNA was isolated from M. ruber and E coli using the Promega Wizard SV Genomic DNA isolation kit protocol. DNA was quantified by spectrophotometry. Universal 16SrRNA primers (IDT, Coralville, IA) were used to amplify the 16SrRNA genes following the supplier’s protocol (Qiagen PCR Master Mix). Question 3. A standard cloning protocol was used to insert an M. ruber PCR product flanked with EcoRIand BamHI restriction sites into the pUC18 plasmid vector. 5 Putative recombinant clones were confirmed by PCR, EcoRI and BamHI digestion and DNA sequencing (DNA Facility (Iowa City, IA). 1.2 *The “unknown protein” (coordinates 41858-42160) was originally identified as trichohyalin . 1.0 0.5 0.3 0.2 Fig. 4: 16SrRNA genes amplified from M. ruber and E coli gDNA using universal primers. Panel A (1% agarose, 100mV for 40 min): Ln1=marker; Ln2=M. ruber gDNA; Ln3=E. coli gDNA; Ln5&6=M. ruber 16SrRNA PCR product; Ln7&8=E. coli 16SrRNA PCR product; Ln8=neg. control. Panel B (1.5% agarose, 100mV for 30 min): Ln1= undigested E. coli 16SrRNA PCR product; Ln2-100bp ladder; Ln3=undigested M. ruber 16SrRNA PCR product; Ln4=E. coli HindIII-digested 16SrRNA PCR product; Ln5=M. ruber HindIII-digested16SrRNA PCR product. Fig. 1: Paralogs were identified for 9 of 23 putative genes in M. ruber. Segments of the M. ruber chromosome are shown using the ”chromosome viewer colored by COG” display from the DOE/JGI’s bioinformatics platform IMG/edu. Putative paralogs were identified for chromate transporter (1), GCN5-related N-acetyltransferase (2), rhodanese-related sulfurtransferase (3), peptidase M23 (4), and succinate-semialdehyde dehydrogenase (5). Not shown are paralogs for N-acetyl-ornithine/N-acetyl-lysine deacetylase, ABC-type branched-chain amino acid transport system, periplasmic component , N-carbamoylputrescineamidase, and phosphoglyceratemutase. 1 2 5 4 5 For further information Please contact Dr. Lori Scott at loriscott@augustana.edu for more information about the Meiothermus ruber Genome Annotation Project and the Microbial Genome Annotation Network. This project is a partnership with the Department of Energy/Joint Genome Institute’s Adopt-a-Genome and Genome Encyclopedia of Bacteria and Archaea (GEBA) project. For more information, about the GEBA project contact Dr. Cheryl Kerfeld, (Education/Structural Genomics Division, Joint Genome Institute) at CKerfeld@lbl.gov. 1 4 4 Fig.5: Cloning M. ruber chromate transporter (A) and N-carbamoylputrescineamidase (B) into pUC18. 1% agarose gels, 100mV for 40min. Panel A: Ln1=marker; Ln2=EcoRI/BamHI -digested clone; Ln4=undigested pUC18; Ln5=digested pUC18; Ln6=undigested putative clone. Panel B: Ln1=marker; Ln2&4=EcoRI/BamHI-digested clones; Ln3=digested pUC18; Ln5=EcoRI-digested clone; Ln6=undigested putative clone; Ln 7=undigested pUC18; Ln8=EcoRI-digested pUC18. The putative clones were confirmed by PCR analysis and DNA sequencing (data not shown). 4 3