Transcript The Fun Part: Testing Hypotheses Generated by the Complete

A Functional Genomics Clearinghouse as an Outcome of Undergraduate
& High School Education – An Update of Efforts at Hiram College
Hiram
genomics
academy
Brad Goodner1, Cathy Wheeler1, Prudy Hall1, Stuart Gordon1, Kathryn Reynolds1, Stephanie Lammlein2, Lindsey Wilson1,
1
1
1
Garrett Temkiewicz , 2002-2007 Molecular & Cellular Biology courses , 2002-2007 Genetics courses , 2006-2007 Introductory
2
3
Biology courses , & 2006-2007 Hiram Genomics Academy sessions
College, Hiram, OH ([email protected], 330-569-5260), 2 Rootstown High School, Rootstown, OH,
& 3 81 high school students from all over Ohio, Michigan, Pennsylvania, & Indiana
Summary of Accomplishments Up to Date
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.
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
2006 Molecular & Cellular Biology Course – 24 Two-Component
System Mutants, 12 Environmental Variables, 54 Treatments
2007 Molecular & Cellular Biology Course – Penicillin Binding Proteins
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.
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 (PBP’s) 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 PBP’s construct new peptidoglycan
chains and crosslink them. Class B PBP’s 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 PBP’s modify peptidoglycan chains before or after crosslinking. Finally, cell walllocalized b-lactamases, besides their impact on antibiotic resistance, can be involved in peptidoglycan
turnover.
Mutant Strain
Atu0050- (actR)
Atu0114- (dctD)
Atu0343- (barA)
Atu0527Atu0629Atu0970- (phoP)
Atu0978- (ragA)
Atu1116- (nwsB)
Atu1297- (pleD)
Atu1446- (ntrC)
Atu1448- (ntrX)
Atu1900- (agp1)
Atu2165- (agp2)
Atu2206Atu2332Atu2434Atu3907Atu4047Atu4300-
Table 1. Data on PBP- mutants obtained by 2007 Molecular & Cellular Biology course.
Class B PBP’s (have only transpeptidase activity):
Atu1067
PBP
PBP2/3
2,8,9
Atu2100
PBP2
PBP3
2,4,8,9
N
N
N
N
N
N
Low MW PBP’s (have either DD-carboxypeptidase or endopeptidase activity):
Atu1499
Dac
PBP5/6
4,5,6,7
N
Atu1505
Dac
PBP5/6
2,6,10
N
Atu2321
Dac
PBP5/6
2,6,10
Y?
Atu3634
DacF
PBP5/6
2,6
Y?
b-Lactamases:
Atu2513
PBP
b-lactamase
11
* 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
N
Growth
Defect
Impact on
Motility
N
N
Very slow
N
Hypomotile
N
Hypermotile
Hypomotile
N
no data
(petR)
(ctrA)
(nasT)
(cvgS)
(nwsB)
Atu4805-
Condition*
glucose-low, glucose, sucrose, xylose, succinate, ammonium-low,
nitrate, glutamine, arginine, 1%NaCl, 2%NaCl, 1.5%KCl, 2%NaCl,
3%KCl, Mg-both5.5, Mg-low7, temp-35C, pH10, Ca-all
succinate, 2%NaCl, Mg-both5.5, pH5.5, Ca-0.1mM,
Ca-none,0.1uM,10mM
succinate,
glucose-low, succinate, 2%lactate
pH5.5
succinate, 3%KCl, Mg-both5.5, pH5.5, temp-15C
glucose-low, Mg-both5.5, pH5.5, Fe-DIP
glucose-low, succinate, Mg-high5.5, pH5.5
proline, 2%NaCl
glucose-low, succinate, serine(C), 2%NaCl, 3%KCl, Fe-M9
succinate, 2%NaCl, pH5.5
glucose, glucose-low, sucrose, xylose, glycerol, succinate,
histidine(C), serine(C), ammonium-low, nitrate, proline,
1%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both7, 1%lactate, 2%lactate,
1%urea, 2%urea, temp-all, pH5.5, phosphate-all, Ca-all, Fe-all, H2O2,
Cu
glucose, glucose-low, sucrose, xylose, glycerol, succinate,
histidine(C), serine(C), ammonium-low, nitrate, serine, proline,
1%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both7, 1%lactate, 2%lactate,
1%urea, 2%urea, temp-all, pHall, pH10, phosphate-all, Fe-all, H2O2,
Cu, Oxyrase, anaerobic
1.5%KCl, 3%KCl
succinate
succinate, 2%NaCl, Mg-high5.5
2%NaCl, temp-15C
succinate, proline, Mg-both5.5, pH5.5
glucose, sucrose, xylose, histidine(C), serine(C), nitrate, 1%NaCl,
2%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both5.5, Mg-low7, 1%lactate,
2%lactate, 1%urea, 2%urea, pH10
xylose, 2%lactate, 2%urea, Ca-10mM
HCA41
HCA61
0.1
0.01
HCA82
0.1
1
10
Figure 3. Atu0512- mutant is
base-sensitive. Growth curve
of Atu0512- mutant in LB broth
at different pH levels.
0.1
5.5
7
8.5
10
0.01
0
10
20
30
40
50
60
70
80
Time (Hrs)
A
Screen for Auxotrophy:
identified steps in pathways for aspartate, branched chain amino acid, histidine,
methioinine, purine, pyrimidine, serine & glycine, & tryptophan synthesis
mutations in glucose dehydrogenase & transaldolase point out crucial role for EntnerDoudoroff & 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
Atu1456 Atu1457
B
Atu1456 Atu1457
N
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).
A
B
100
1
Very hypermotile
Hypomotile
N
HCA40
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
* Interpretation of impact results:
blue means the mutant showed growth >50% greater than that seen for wt
red means the mutant showed >50% 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)
Hypomotile
no data
Slow
N
Wildtype
1
[Pi] in ATmannitol (mM)
The Fall 2007 Molecular & Cellular Biology course at Hiram College (50 students) set out to look at
the function of the 11 PBP’s encoded by the A. tumefaciens C58 genome. During the course of a 12week 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 Atu1341mutant had a dramatic cell shape change and showed a large decrease in growth rate. The Atu1341cells 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.
Possibly
Essential
B
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 C 100
Atu4668 on ChrII
10
encodes a ABC transporter permease
Figure 2. Biofilm defective mutants. A: Colony morphology of selected
mutants on LB-NaCl+dyes. 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.
E. coli and Bacillus subtilis have been the models for studying PBP activities. Data from these two
organisms have shown that the Class B PBP’s 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
PBP’s influence the uniformity of cell shape by altering the cross-linking level and substrate
availability.
Gene
Current
Best Hit
Domains identified
Number
Annotation
to E. coli
by InterProScan*
Class A PBP’s (have both glycosyltransferase & transpeptidase activities):
Atu0103
PBP1A
PBP1B
1,2,3,4,14
Atu0931
PBP
PBP1B
1,2,3,4
Atu1341
PBP1A
PBP1A
1,2,3,4
Atu3694
PBP
PBP1C
1,2,4,12,13
Interesting Stories from Forward Genetics
A
Adhered/Planktonic Ratio
Abstract
Optical Density at 595 nm
1 Hiram
C
D
E
F
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 betaProteobacteria, respectively. Atu1456 CysI ortholog shown in pale green while
Atu1457 ortholog shown in bright red.
This work was funded by Hiram College and by