Download Functional Uniqueness Hiding Under Redundancy of Sucrose

Genomics as a Means,
Not an End, to
Understanding the
Biology of a “Simple”
Soil Bacterium
Brad Goodner
Department of Biology
Hiram College, Hiram, OH
Teaching
Research
Agrobacterium
bv. 2 & 3 strains
(NSF grant w/ 7 partners)
2 Xenorhabdus species
(USDA grant w/ 6 partners)
Azotobacter vinelandii
(NSF grant w/ 4 partners)
Collaborations
Hiram
Genomics
Initiative
Chromohalobacter salexigens
(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea
(w/ Monsanto Co.)
Hiram
Students
High
school
Students
Recruiting
Hiram Genomics Initiative
Agrobacterium
Genome Project
Other Genome Projects
Sphingomonas Chromohalobacter
Xenorhabdus
Azotobacter
elodea
salexigens bovienii & nematophila vinelandii
Functional Genomes of
Native Genomics of K84 (bv. 2)
Tumor Strain C58 & S4 (bv. 3)
Genetic/
Survey (biovar 1)
Physical Map
(high
schools)
(Genetics)
Genetic/
Physical Map
Genetic/
Physical Map
(Genetics)
(Genetics &
high schools)
Gap
Closure
(Independent
Research)
Sequence
Annotation
(MolCell, Genetics,
& Biochem)
Gene
Disruptions
(MolCell &
Independent
Research)
Mutant
Gap
Screens Closure
Sequence
Annotation
(MolCell & (Independent (Genetics &
Independent Research) Independent
Research)
Research)
Gap
Closure
Genetic/
Physical Map
(Independent (Genetics &
Research)
Independent
Research)
Sequence
Annotation
(Independent
Research)
Using Research to Bridge
Teaching-Learning Gaps
Within Courses
• What prevents us from
incorporating original
research into the lab
component of courses?
• Must excite students – move into
independent research projects
• Must excite us
• Must teach key skills & concepts
• Must be doable within time, space,
& budget constraints
• Must be successful as measured
by the norms of science –
effective training for the future,
presentations at conferences, &
publications
Basics of a Genome Project
Genome
6-8X
Sequencing
Coverage
Overlaps in
Small Pieces
to Form Contigs
Gap Closure
Random
Pieces
Shotgun
Genomic
Libraries
Join Large
Pieces into
Sequenced
Genome
Annotation
Functional Genomics
Genetic/
Physical
Map
Annotation of
Contig Ends
Example of Success:
Agrobacterium Genome Project
bacterium
hormones
DNA
plant cell
food
• Has involved >300 students within course
research projects as well as in independent
projects (at Hiram College & University of
Richmond) since 1996
• 19 student authors on publications in
Journal of Bacteriology & Science
• >50 student authors on >30 posters
presented at research conferences
• Successful involvement in collaborations
with companies & larger universities
Examples I Will Cover Today
• 1) Sequence annotation by >70 students in MolCell &
Genetics courses
• 2) Sucrose metabolism (Jen Hardesty, Mandy Reed, Ginny
Mateo)
• 3) Aconitases (DaJuan Whiteside, Terrence Johnson,
Razan Yasin, Gina Dottle, John Mark Kuhns, Torrie Ohlin,
Telisha Law)
• 4) Selenite tolerance & reduction (Frank Arnold, Dan
Arnold, Josh Collins)
Bioinformatic Analysis
of Pathways in C58
(bv1) & S4 (bv3)
Part I = Identify players in pathway & note
any absences or redundancies
12 proteins for glycolysis & gluconeogenesis
Avi5336 is most likely glucokinase
Avi0235 is likely phosphoglycerate mutase
role of phosphofructoskinase (pfk) is
apparently performed by pyrophosphate-fructose-6-phosphate 1-phosphotransferase
(fbp)
Agrobacterium has never been shown to
have fructose 1,6-bisphosphatase and it was
absent in both biovars
Redundancies
gpm
glk
pyk
pck
C58
S4
ChrI Chr2 Chr1 Chr2
1 1
2
1
1
1
1
2
1
2
Bioinformatic Analysis
of Pathways in C58
(bv1) & S4 (bv3)
Part II – Identify potential operons
There was one potential operon in both
biovars. They both involved a
phosphoglycerate kinase gene and a fructose
bisphosphate aldolase gene. In this instance,
the genes in the operon were on ChrI of S4
but were on ChrII of the C58 genome
Part III – Identify potential lateral gene
transfer events
None of our genes appear to have arrived in
the genome via lateral gene transfer
Functional Uniqueness
Hiding Under Redundancy
of Sucrose Metabolism
• Sucrose is major transportable
form of organic carbon in plants
• Role of sucrose metabolism by
Agrobacterium in interactions
with plants before & after
tumorigenesis is unknown
• Failure of standard genetic
approach to find sucrosenonutilizing mutants
• Enzymatic work of 60’s & 70’s
suggest at least 2 routes for
sucrose degradation
• Genome sequence shows that
the situation is much more
complicated
Putative Sucrose Metabolic Routes
3GDH
Sucrose
3-keto-sucrose
?
Agl
transporter
3-keto-sucrose
OM
?
PM
Sucrose
?
3-keto- fructose
a-glc II
glucose
a-glc I
Sucrose
hydrolase
?
glucose
fructose + glucose
Growth Assays on
Single Gene Knockouts
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
C58 suc hyd- growth on M9 minimal
0.5
0.4
Glucose
Sucrose
OD600
OD600
C58 wildtype growth in M9 minimal
0.3
Glucose
Sucrose
0.2
0.1
0
1
2
3
4
5
6
7
Time (hr)
8
9 10 11
1
2
3
4
5
6
7
8
9
Time (hr)
Growth ratio compared to glucose in M9 minimal:
Strain
sucrose trehalose raffinose cellobiosepalatinosemaltulose
C58
1.25
1.19
1.13
0.84
1.08
1.25
C58 suc hyd0.71
0.77
0.91
0.71
0.53
0.95
C58 glcI0.85
0.84
0.87
0.69
0.76
0.84
C58 glcII0.78
0.72
0.86
0.65
0.71
0.71
Osmotic Stress Assays
Growth in M9glucose + 1 mM sucrose:
Strain
Water 0.1M NaCl 0.5M NaCl
C58
1
1
0.77
C58 suc hyd1
0.02
0
C58 glcI1
0.37
0.01
C58 glcII1
0.63
0.01
Strain
C58
C58 suc hyd-
M9glucose+1 mM
M9glucose+1 mM
M9glucose+1 mM
M9glucose+1 mM
sucrose:
trehalose:
glutamate:
betaine:
0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl
1
0.77
0.88
1.24
0.95
0.58
0.91
0.48
0.02
0
1.01
0.04
1
0.07
0.28
0
Osmotic Adjustment
in A. tumefaciens
Smith et al., 1990, J. Bact. 172:6849-55
One Enzyme – Multiple Roles?
Or
A Different Role Is All There Is?
Glucose + Fructose
Sucrose
sucrose hydrolase
Mannose
Mannosucrose
Glucose
Glc-I
?
Suc hyd
Sucrose
metabolism
Glc-II
?
Osmotic
stress
Other Genes Involved in
Osmotic Adjustment
wildtype
Atu3741MFS-type sugar permease
Atu4610Sugar nucleotide epimerase
Atu1588Homoserine dehydrogenase
Aconitases
Many bacteria have 2
Have roles beyond
the TCA Cycle
Agrobacterium bv1 is
exceptional within
a-Proteobacteria to
have >1 aconitase
Pfu
HamA
RfeA
CpeA
CbuA
IloA
PflA
AviA
SenA
EcoA
SilA
RspA
CcrA
BquA
MesA
BsuA
SmeA
AtuA
AvtA
BjaA
RpsA
EruA
WolA
ParA
Mav
KraA
ArtA
DgeA
Bcr
Sha
Sau
BceA
AdeA
SacA
FacA
XcaA
NmeA
Dvu
Hma
AtuB
SilB
AdeB
SynB
NmeB
MmaB
BceB
XcaB
ParB
IloB
PflB
AviB
SenB
EcoB
a
a
g
a
a
AcnA’s
Actinobacteria
Firmicutes
b
d
g
b
d
a
d
b
a
b
g
AcnB’s
g
E. coli Model for
Aconitase Functions
• AcnB is major TCA isoform
• AcnA is induced during stationary phase
• Acn’s lose Fe-S center during Fe starvation or oxidative stress
& act as RNA-binding apo-proteins … impact gene expression
 DAcnB leads to hypomotility, while DAcnA has normal motility
Agrobacterium C58
Aconitase Gene
Knockouts
AcnA is Major Player!
5
Colony Swarming (cm diameter)
4.5
4
Hour 0
Hour 6.5
Hour 24
Hour 30.5
3.5
3
2.5
2
1.5
1
0.5
0
C58
(LB
0.3)
C58
(LB
0.6)
C58
(LB 1)
C58
(LB
1.5)
acnA(LB
0.3)
acnA(LB
0.6)
Strain
(M edium, % Agar)
acnA(LB 1)
acnA(LB
1.5)
Agrobacterium C58
Aconitase Gene
Knockouts
AcnA is Major Player!
wildtype
AcnA-
Agrobacterium C58
Aconitase Gene Knockouts
AcnA is Major Player!
Weird Pigmentation on
Certain Media (Contains Selenite)
UK1
C58
Schroth Minimal Medium
Pigmentation Due to
Strange Globules (Reduced Se?)
Strain FL
(Silver Creek, OH)
Strain UK1
(stream bank in UK)
Pigmentation Due to Reduction
of Selenite to Elemental Se
Strain C58
Strain S4
Strain UK1
Mutants Impacted in
Response to Selenite
Mutant
C58sel1
C58sel6
C58sel9
Site of Tn Insertion
Atu0238 thioredoxin reductase family member
Atu3466 ArsR TF family member
Atu0284 TspO (regulates response to many stresses)
Mutants Impaired in Response to Selenite
2.5
C58 LB
C58Sel1 LB
C58Sel4 LB
Absorbance (600nm)
2
C58Sel6 LB
C58Sel9 LB
C58 Se
1.5
C58Sel1 Se
C58Sel4 Se
C58Sel6 Se
1
C58Sel9 Se
0.5
0
0
10
20
30
Time (Hours)
40
50
60
Teaching
Research
Agrobacterium
bv. 2 & 3 strains
(NSF grant w/ 7 partners)
2 Xenorhabdus species
(USDA grant w/ 6 partners)
Azotobacter vinelandii
(NSF grant w/ 4 partners)
Collaborations
Hiram
Genomics
Initiative
Chromohalobacter salexigens
(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea
(w/ Monsanto Co.)
Hiram
Students
High
school
Students
Recruiting
Bioinformatic Hunt for
3-Ketosucrose Pathway
Inferences from Literature:
• Pathway is not found in
Sinorhizobium, Mesorhizobium, & Brucella
• G3DH is FAD-dependent
dehydrogenase
• G3DH is periplasmic
• G3DH gene near genes for
associated chemo-taxis,
transport, ETC, hydrolysis,
& reduction enzymes
Bioinformatic Analysis of C58:
• Used TIGR Comprehensive
Microbial Genome Database to
identify dehydrogenases
unique to Agrobacterium
• 2 appeared to be FADdependent
• 1 appeared periplasmic
• Nearby genes for MCP,
cytochrome, & reductase
• Mutant is still G3DH+!
• Now have G3DH- mutants from
large-scale random screen …
implicate 2 separate ABC-type
sugar transport systems
Diversity of Responses
to Selenite
A. C58
Cell Density (OD600)
1.000
0.100
0.010
0
10
20
30
40
50
60
Time (hours)
B. UK1
Cell Density (OD600)
1.000
0.100
0.010
0
10
20
30
Time (hours)
40
50
60