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,
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Elizabeth , Lehpamer, Nicole , Oldfather, Nicole , Allibone, Kevin and Sollenberger, Ryan
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Introduction
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:
1. 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?
2. Could high quality genomic DNA be isolated from M. ruber that was
suitable for PCR?
3. Could select M. ruber genes be cloned into the pUC18 plasmid vector and
transformed into E. coli for future functional genomics studies?
College, Rock Island, IL.
2Scott
Results
Table 1: Bioinformatics analysis supported the original gene-call for 22 of the 23
ORFs studied within the M. ruber genome
Community College/EICCD, Bettendorf, IA
Conclusions
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.
Fig. 6. The Inquiry Wheel, a
contemporary view of the
scientific process.8 Questioning
is placed as the center.
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.
Firmicutes
Proteobacteria
Deinococcus-Thermus
Cyanobacteria
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 GCN5related N-acetyltransferase gene (data not shown). Two M. ruber genes
were confirmed as cloned into the pUC18 (chromate transporter and Ncarbamoylputrescine amidase) by restriction enzyme analysis ,and by PCR
and sequencing (data not shown).
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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.
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.
Literature cited
Materials and methods
Question 1. Twenty-three M. ruber genes were annotated using the online IMGACT/edu bioinformatics toolbox created by the DOE JGI.4 The annotation
process is organized by module and uses the following bioinformatics tools:
*The “unknown protein” (coordinates 41858-42160) was originally identified as
trichohyalin .
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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 EcoRI and 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).
DOE 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).
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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 succinatesemialdehyde dehydrogenase (5).
Not shown are paralogs for Nacetyl-ornithine/N-acetyl-lysine
deacetylase, ABC-type branchedchain amino acid transport system,
periplasmic component , Ncarbamoylputrescine amidase, and
phosphoglycerate mutase.
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-digested 16SrRNA PCR product.
For further information
Fig.5: Cloning M. ruber chromate transporter (A) and N-carbamoylputrescine amidase (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).
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.