A Functional Genomics Clearinghouse as an Outcome of Undergraduate

1
A Functional Genomics Clearinghouse as an Outcome
of Undergraduate High School Education An
Update of Efforts at Hiram College Brad
Goodner1, Cathy Wheeler1, Prudy Hall1, Stuart
Gordon1, Kathryn Reynolds1, Stephanie Lammlein2,
Lindsey Wilson1, Garrett Temkiewicz1, 2002-2007
Molecular Cellular Biology courses1, 2002-2007
Genetics courses1, 2006-2007 Introductory
Biology courses2, 2006-2007 Hiram Genomics
Academy sessions31 Hiram College, Hiram, OH
(goodner_at_hiram.edu, 330-569-5260), 2 Rootstown
High School, Rootstown, OH, 3 81 high school
students from all over Ohio, Michigan,
Pennsylvania, Indiana
Hiram genomics academy
Abstract Obtaining the complete genome sequence
of any organism is really just a new beginning
you have a lot of tools now available but are not
yet sure how best to use them. Functional
genomics is really just an extension of how
molecular biology and microbiology have made
progress over the past 25 years or more through
characterization of mutants through forward or
reverse genetics. The extension is one of
breadth to include as many genes as possible.
Over the past 6 years, Hiram College faculty and
students have accumulated a large set of
mutations in the A. tumefaciens C58 genome (and
to a lesser extent in the A. rhizogenes A4
genome). Students in the Molecular Cellular
Biology course use reverse genetics to test
functional predictions based on bioinformatics
analyses of gene function. We will highlight the
work done by the 2006 and 2007 iterations of the
course. Students in the Genetics course, along
with high school students during the past two
years, have used forward genetics to link genes
to functions through a large scale mutant hunt.
We will highlight some of the more interesting
findings in several functional categories.
Finally, we will look forward to future efforts
and we are certainly open to new suggestions from
others. 2007 Molecular Cellular Biology Course
Penicillin Binding Proteins The bacterial
cell wall is crucial for withstanding turgor
pressure and also as a cell shape determinant.
The wall is composed of a heteropolymer called
peptidoglycan that has both polysaccharide and
peptide characteristics. The major enzymes
involved in wall synthesis, maintenance, and
turnover are nicknamed Penicillin-Binding
Proteins (PBPs) because the antibiotic
penicillin and its derivatives act as
irreversible enzyme poisons on these proteins.
Most rod-shaped bacteria have 6 or more PBPs that
fall into 4 categories. Class A PBPs construct
new peptidoglycan chains and crosslink them.
Class B PBPs are only involved in cros-linking
peptidoglycan chains, but do so in either a
elongation-growth or septation-growth mode
depending on the enzyme. The low molecular
weight PBPs modify peptidoglycan chains before
or after crosslinking. Finally, cell
wall-localized b-lactamases, besides their impact
on antibiotic resistance, can be involved in
peptidoglycan turnover. E. coli and Bacillus
subtilis have been the models for studying PBP
activities. Data from these two organisms have
shown that the Class B PBPs influence cell shape
in terms of the lengths of rods or rod-to-coccus
transitions because of their differential
activities during the cell cycle, and the low MW
PBPs influence the uniformity of cell shape by
altering the cross-linking level and substrate
availability. The Fall 2007 Molecular Cellular
Biology course at Hiram College (50 students) set
out to look at the function of the 11 PBPs
encoded by the A. tumefaciens C58 genome. During
the course of a 12-week semester, the students
were able to make mutations in 9 of the 11 genes
and to obtain phenotypic data on 8 of the
mutants. Here are their initial observations
(also see Table 1) While some of the mutants
might have a slight change in cell shape, the
Atu1341- mutant had a dramatic cell shape change
and showed a large decrease in growth rate. The
Atu1341- cells had a variety of amorphous shapes
with few regular rods. Most of the mutants
had a definite impact on cell motility. The
Atu1499- mutant is very hypermotile, as compared
to wildtype, on both 0.3 and 0.9 agar plates.
The Atu1341- mutant is hypermotile only on 0.3
agar. The Atu1067-, Atu1505-, Atu2513-, and
Atu3694- mutants were hypomotile on all agar
concentrations tested.
Interesting Stories from Forward Genetics Screen
for Biofilm Defects (change in colony morphology
on LB agar minus NaCl plus Coomassie Blue and
Congo Red Figure 2) Atu2121 on ChrI encodes
Lyc glycosyl hydrolase may be involved in
exopolysaccharide synthesis or maintenance Atu2660
on ChrI (HCA61) encodes a conserved
hypothetical protein Atu3327 on ChrII
(HCA41) encodes ExoY succinoglycan
exopolysaccharide synthesis protein Atu3437 on
ChrII encodes a ABC transporter ATP-binding
protein Atu3740 on ChrII (HCA82) encodes
fructose bisphosphate aldolase may influence
substrate availability for making
exopolysaccharide Atu4000 on ChrII
(HCA40) encodes BioA adenosylmethionine-8-amino-
7-oxononanoate aminotransferase role of
biotin in biofilm? in E. coli, bioF- mutant has
altered biofilm Atu4053 on ChrII encodes ExoA
succinoglycan biosynthesis protein Atu4668 on
ChrII encodes a ABC transporter
permease Screen for pH Tolerance (lack of
growth on LB agar pH 5.5 or LB 10) 15 acid
sensitive mutants all of them only show defect
on solid medium! Why? 4 base sensitive mutants
(Figure 3) most interesting is Atu0512 encoding
PhaA pH adaptation K efflux system
component mutation in Atu0512 homolog of
Sinorhizobium is base-sensitive is
nodulation-negative experiments underway to see
if Atu0152- mutant is virulent Screen
for Auxotrophy identified steps in pathways
for aspartate, branched chain amino acid,
histidine, methioinine, purine, pyrimidine,
serine glycine, tryptophan synthesis mutatio
ns in glucose dehydrogenase transaldolase point
out crucial role for Entner- Doudoroff
pentose phosphate pathways mutation in glycogen
synthase impacts use of glucose but not of
glycerol mutation in Atu3885 encodes a inositol
monophosphatase family member that is
needed for growth on both glucose and
glycerol role? mutation in Atu1457 leads to
auxotrophy for sulfur gene encodes a conserved
hypothetical protein in many organisms,
this gene is right next to Atu1456 encoding
CysI sulfite reductase hemoprotein beta subunit
(Figure 4) our working hypothesis is that
Atu1457 encodes a previously unknown protein of
the cysteine synthesis pathway students at
Rootstown High School are currently working to
see if the auxotrophic phenotype in Atu1457-
mutant is due to that mutation or just a
polar effect within the operon
Summary of Accomplishments Up to Date Reverse
Genetics 75 specific genes disrupted so far
mutant phenotypes characterized Forward
Genetics 10,000 Agrobacterium transposon
insertion mutants generated 10,000 mutants
screened for 10 different phenotypes 160
mutants with interesting phenotypes saved for
further analysis genomic DNA isolated from 119
of these mutants the site of transposon insertion
cloned out sequenced
A
B
2006 Molecular Cellular Biology Course 24
Two-Component System Mutants, 12 Environmental
Variables, 54 Treatments Two-component systems
are a common regulatory mechanism in bacteria and
the A. tumefaciens genome is loaded with 34
paired teams, 5 hybrid proteins, 17 orphan
histidine sensor kinases, and 16 orphan response
regulators. Previous work by others had
characterized 7 proteins that fall into this
family. The 2005 and 2006 iterations of the
MolCell course knocked out 11 and 16,
respectively, of the response regulator genes.
The 2006 course used a new microtiter plate
reader to study the phenotype of 20 of the
response regulator mutants. A summary of their
findings is given in Table 2.
C
Figure 2. Biofilm defective mutants. A Colony
morphology of selected mutants on LB-NaCldyes.
Wildtype is shown at the top. B Pellicle
morphology of selected mutants. Wildtype is
shown at the top. C Assay for binding to
polystyrene microtiter wells under different Pi.
Table 2. Summary results for a phenotypic
microarray of 20 response regulator
mutants. Mutant Strain Condition Atu0050-
(actR) glucose-low, glucose, sucrose, xylose,
succinate, ammonium-low, nitrate, glutamine,
arginine, 1NaCl, 2NaCl, 1.5KCl, 2NaCl,
3KCl, Mg-both5.5, Mg-low7, temp-35C, pH10,
Ca-all Atu0114- (dctD) succinate, 2NaCl,
Mg-both5.5, pH5.5, Ca-0.1mM, Ca-none,0.1uM,10m
M Atu0343- (barA) succinate, Atu0527-
glucose-low, succinate, 2lactate
Atu0629- pH5.5 Atu0970- (phoP) succinate,
3KCl, Mg-both5.5, pH5.5, temp-15C Atu0978-
(ragA) glucose-low, Mg-both5.5, pH5.5,
Fe-DIP Atu1116- (nwsB) glucose-low, succinate,
Mg-high5.5, pH5.5 Atu1297- (pleD) proline,
2NaCl Atu1446- (ntrC) glucose-low, succinate,
serine(C), 2NaCl, 3KCl, Fe-M9 Atu1448-
(ntrX) succinate, 2NaCl, pH5.5 Atu1900-
(agp1) glucose, glucose-low, sucrose, xylose,
glycerol, succinate, histidine(C), serine(C),
ammonium-low, nitrate, proline, 1NaCl,
1.5KCl, 2NaCl, 3KCl, Mg-both7, 1lactate,
2lactate, 1urea, 2urea, temp-all, pH5.5,
phosphate-all, Ca-all, Fe-all, H2O2, Cu Atu2165
- (agp2) glucose, glucose-low, sucrose, xylose,
glycerol, succinate, histidine(C), serine(C),
ammonium-low, nitrate, serine, proline, 1NaCl,
1.5KCl, 2NaCl, 3KCl, Mg-both7, 1lactate,
2lactate, 1urea, 2urea, temp-all, pHall,
pH10, phosphate-all, Fe-all, H2O2, Cu,
Oxyrase, anaerobic Atu2206- (petR) 1.5KCl,
3KCl Atu2332- succinate Atu2434-
(ctrA) succinate, 2NaCl, Mg-high5.5 Atu3907-
(nasT) 2NaCl, temp-15C Atu4047-
(cvgS) succinate, proline, Mg-both5.5,
pH5.5 Atu4300- (nwsB) glucose, sucrose, xylose,
histidine(C), serine(C), nitrate, 1NaCl,
2NaCl, 1.5KCl, 2NaCl, 3KCl, Mg-both5.5,
Mg-low7, 1lactate, 2lactate, 1urea,
2urea, pH10 Atu4805- xylose, 2lactate,
2urea, Ca-10mM Interpretation of impact
results blue means the mutant showed growth
gt50 greater than that seen for wt red means the
mutant showed gt50 less growth than that seen for
wt bold means in a ratio to wt under that
condition (e.g., mutant at pH5.5/wt at
pH5.5) underlined means as a ratio to itself at
baseline condition (mutant pH5.5/mutant pH7
compared to wt 5.5/wt 7)
Figure 3. Atu0512- mutant is base-sensitive.
Growth curve of Atu0512- mutant in LB broth at
different pH levels.
Table 1. Data on PBP- mutants obtained by 2007
Molecular Cellular Biology course. Gene Curren
t Best Hit Domains identified Possibly
Growth Impact on Number Annotation to E.
coli by InterProScan Essential
Defect Motility Class A PBPs (have both
glycosyltransferase transpeptidase
activities) Atu0103 PBP1A PBP1B 1,2,3,4,14 N
N Hypomotile Atu0931 PBP PBP1B 1,2,3,4 N N
N Atu1341 PBP1A PBP1A 1,2,3,4 N Very
slow Hypermotile Atu3694 PBP PBP1C 1,2,4,12,13
N N Hypomotile Class B PBPs (have only
transpeptidase activity) Atu1067 PBP PBP2/3 2,
8,9 N N Hypomotile Atu2100 PBP2 PBP3 2,4,8,9
N no data no data Low MW PBPs (have
either DD-carboxypeptidase or endopeptidase
activity) Atu1499 Dac PBP5/6 4,5,6,7 N
Slow Very hypermotile Atu1505 Dac PBP5/6 2,
6,10 N N Hypomotile Atu2321 Dac PBP5/6 2,6,1
0 Y? Atu3634 DacF PBP5/6 2,6 Y? b-Lactamas
es Atu2513 PBP b-lactamase 11 N N N
InterProScan Domains 1 Family 51 glycosyl
transferase domain 2 PBP transpeptidase
domain 3 PBP1a domain 4 Beta-lactamase
transpeptidase domain 5 Peptidase S13
D-Ala-D-Ala carboxypeptidase C domains 6
Peptidase S11 D-Ala-D-Ala carboxypeptidase A
domains 7 PBP-associated domain 8 Class A
Beta-lactamase 9 PBP dimerization domain 10
Cell division protein domain 11 Beta-lactamase
family 12 PBP1c domain 13 PBP C-terminal
domain 14 WW/Rs5/WWP domain
Atu1456 Atu1457
Atu1456 Atu1457
B
A
C
Figure 1. Cell shape of the Atu 1341- mutant is
highly altered. A, B Atu1341- mutant. C, D
Atu1499- mutant. E, F Atu2513- mutant. A, C,
E Light microscopy of crystal violet-stained
cells. B, D, F Fluorescent confocal microscopy
of Bocillin FL-labeled cells (fluorescent
penicillin derivative).
Figure 4. Atu1457 orthologs are in a consistent
genomic context. A Position of the Atu1457
ortholog (pale green arrow) just behind Atu1456
CysI ortholog (bright red arrow) in a wide
variety of genera within the alpha-Proteobacteria.
B, C Similar alignment for members of the
gamma-Proteobacteria and beta-Proteobacteria,
respectively. Atu1456 CysI ortholog shown in
pale green while Atu1457 ortholog shown in bright
red. This work was funded by Hiram College
and by
A
B
F
E
D
C