Download Linked Genes

FGAR Amidotransferase: A Purine
Biosynthetic Enzyme with Potential
as a Target for Antimicrobial Drugs.
Jeffrey D. Newman
Lycoming College
May 7, 1998
Overview
• Symbiosis Project
– Background - Graduate work
– Goal - Identification of genes associated with
commitment to determinate nodule development.
• Purine Project
–
–
–
–
–
–
5/23/2017
Historical Background
Purine Auxotroph Phenotypes - The Problem
Identification of Purine Genes - The Hypothesis
Support from Genome Sequences
Recent Results - Heterologous complementation
Future Directions
Slide #2
Rhizobium-Legume Symbiosis
Early Steps
Attachment, nod gene induction
Root hair curling, meristem initiation
Infection thread development
5/23/2017
Slide #3
Rhizobium-Legume Symbiosis
Intermediate Steps
Infection thread development
Nodule Emergence
and Release from
Infection Thread.
5/23/2017
Slide #4
Symbiotic phenotype of
Rhizobium purine auxotrophs
• Purine auxotrophs initiate nodule
formation (Nod+), but do not
induce infection thread
development (Inf -).
Pur - + purine
• Lack of infection leads to
formation of pseudonodules.
Pur - + AICAr
• Addition of AICA riboside but not
purines restores infection and
enhances nodule development.
5/23/2017
WT
Slide #5
The Purine Biosynthetic Pathway
TPP
purF purD purN purLQ purM
PRPP
AIR
AICAR
GMP
PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate
AIR = 5-aminoimidazole ribonucleotide
AICAR = 5-aminoimidazole-4-carboxamide
ribonucleotide
TPP = Thiamine Pyrophosphate
5/23/2017
IMP
AMP
Slide #6
Hypotheses
• Hypothesis 1: A derivative of
AICAR serves as signal for
infection thread development
NH2
NH2
C
N
N
5/23/2017
N
H2N
N
O
N
N
• AICA riboside must be present
until day 6 for true nodule
development (Ndv+).
• Hypothesis 2: Infection through
day 6 triggers nodule-specific
developmental pathway.
C
AMP
RP
AICAR
RP
NH2
C
N
N
N
Cytokinin
N
RP
Slide #7
Symbiosis Research Projects
• Long Term Goal - Identification of genes associated
with commitment to determinate nodule development.
– Use Rhizobium expressing GFP to examine relationship between
AICA riboside supplementation and infection thread development.
– Transition to Rhizobium loti - Lotus model system.
– Use transgenic Lotus to examine promoters induced at the time
of commitment to nodule organogenesis.
5/23/2017
Slide #8
The Purine Biosynthetic Pathway
TPP
purF purD purN purLQ purM
PRPP
AIR
AICAR
GMP
PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate
AIR = 5-aminoimidazole ribonucleotide
AICAR = 5-aminoimidazole-4-carboxamide
ribonucleotide
TPP = Thiamine Pyrophosphate
5/23/2017
IMP
AMP
Slide #9
An Intriguing Observation
Mutant Strains
Pur?
Thi?
Accum? Explanation
CE106, CE115, CE170,
CE292, CE382
-
-
-
CE124, CE290, CE384
-
+
+
AIR
CE110, CE390, CE391
5/23/2017
-
Blocked before
AIR
Blocked between
AIR and AICAR
???????
Slide #10
Complementation Analysis
5/23/2017
Cosmid
Strains Complemented
pCOS106
CE106
pCOS110
CE110, CE170, CE382,
CE390, CE391
pCOS115
CE115, CE292
pCOS124
CE124, CE290, CE384
Slide #11
Marker Exchange Recovery of Mutations
• In a complemented Rhizobium
strain, some recombination between
plasmid and chromosome will occur.
• Plasmid transferred into E. coli,
selection for Tn5-encoded KanR.
pur gene
• Cosmids containing pur gene
mutations designated pJNXXXA.
– e.g. - CE110  pJN110A
5/23/2017
Slide #12
pCOS110
Complementation Groups
CE110 CE390
pCOS110
pJN110A
pJN390A
pJN391A
pJN170A
pJN382A
5/23/2017
+
+
+
+
+
+
+
+
+
CE391
CE170
CE382
+
+
+
+
+
+
+
+
-
+
+
+
+
Slide #13
pCOS110 Map
• Initial partial sequencing out from Tn5 identified
purY, purQ, and purL mutations.
• Subsequent complete sequencing identified purC
and YJII.
E=EcoRI
B=BamHI
B
Completed sequence
E
E
CE170
CE382
CE110
E
5/23/2017
CE391
1 kb
E
B
Partial sequence
Slide #14
The Purine Biosynthetic Pathway
TPP
purF purD purN purLQ purM
PRPP
AIR
AICAR
GMP
PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate
AIR = 5-aminoimidazole ribonucleotide
AICAR = 5-aminoimidazole-4-carboxamide
ribonucleotide
TPP = Thiamine Pyrophosphate
5/23/2017
IMP
AMP
Slide #15
FGAR Amidotransferase
+H N
3
CH
CH
+H N
3
COO
CH
2
CH 2
NH
2
+
N
N H
O
O
C
H
O
O
O
N H
HN
OH2 C
C
H
O
O
O
OH
OH
5'-phosphoribosyl-N- formylglycinamide
FGAR
5/23/2017
P
H
N
O
O
OH2 C
+
O
glutamate
O
P
2
C
H
glutamine
O
COO
CH 2
C
O
CH
OH
OH
5'-phosphoribosyl-N- formylglycinamidine
FGAM
Slide #16
FGAR Amidotransferase
Organization
Type I
Single subunit
ATP-binding, cleavage
FGAR
glutamine
enolization amide transfer
PurL - 1295 aa (E.coli)
Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Pseudomonas
aeruginosa, Vibrio cholerae, Neisseria gonnorheae, Neisseria meningitidis, Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Caenorabditis elegans, Drosophila melanogaster,
Gallus gallus, Homo sapiens.
Type II
Multi-subunit
PurL - 742 aa (B.subtilis )
PurQ - 227 aa
Rhizobium etli*, Bacillus subtilis*, Lactobacillus caseii, Staphylococcus aureus*,
Mycobacterium tuberculosis*, Mycobacterium leprae*, Streptococcus pneumoniae,
Enterococcus faecaelis*, Synechocystis spp.*, Deinococcus radiodurans*, Thermotoga
maritima, Aquifex aeolicus*, Archaeoglobus fulgidus*, Methanococcus jannaschii*,
Methanobacterium thermoautotrophicum.
5/23/2017
Slide #17
FGAR Amidotransferase
Phylogenetic Analysis
proteobacteria
   

cyanobacteria
plants
Gram +
Methanogens
Deinococci
animals
Extreme
thermophiles
fungi
Thermotoga
Aquifex
5/23/2017
Type I
Type 2
unknown
Slide #18
PurY as a Third Subunit
SalI
EcoRI
EcoRI
E.coli purL
3.9 kb
Mob
CmR
SalI
SalI
pTB93F (8.0 kb)
GFP
SalI
pTrp
from Daniel Gage
of Sharon Long’s
Lab
Rep
EcoRI
SpcR
5/23/2017
Slide #19
PurY as a Third Subunit
Complementation
Strain Mutation by E.coli purL
5/23/2017
CE106
PurF::Tn5
?
CE110
PurY::Tn5
+
CE170
PurL::Tn5
+
CE390
PurQ::Tn5
?
Slide #20
Future Work
• Finish Rhizobium purL sequence.
• Overexpression and purification of
Rhizobium and/or Staphylococcus
aureus PurY, PurQ, PurL and E.coli
PurL polypeptides.
• FGAR Amidotransferase assays.
• Identification of Type 2 inhibitors.
5/23/2017
Slide #21
Lab Members
• Alumni
– Diana Burley
– Kevin Ferguson
– Jonathan Cook
– Kathy Roberts
– Matthew Georgy
5/23/2017
• Current
– Mark McCleland
– Kim Mistiszyn
– Lori Schultz
– Rachel Lawton
– Laura Singer
– Tom Rombold
Slide #22