Transcript 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.
• 4/25/2020
Purine Project
– – – – – –
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
4/25/2020
Infection thread development
Slide #3
Rhizobium-Legume Symbiosis Intermediate Steps
Infection thread development Nodule Emergence and Release from Infection Thread.
Slide #4 4/25/2020
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. 4/25/2020 WT Slide #5
The Purine Biosynthetic Pathway PRPP purF purD purN purLQ purM AIR TPP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate GMP
4/25/2020
AMP IMP AICAR
Slide #6
Hypotheses
• Hypothesis 1: A derivative of AICAR serves as signal for infection thread development
N NH 2 C N N N
• AICA riboside must be present
AMP
until day 6 for true nodule development (Ndv + ).
• Hypothesis 2: Infection through day 6 triggers nodule-specific developmental pathway.
4/25/2020
N RP NH 2 C N Cytokinin O N N RP NH 2 C H 2 N AICAR N 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.
4/25/2020 Slide #8
The Purine Biosynthetic Pathway PRPP purF purD purN purLQ purM AIR TPP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate GMP
4/25/2020
AMP IMP AICAR
Slide #9
An Intriguing Observation
Mutant Strains CE106, CE115, CE170, CE292, CE382 Pur?
CE124, CE290, CE384 CE110, CE390, CE391 Thi?
Accum? Explanation -
Blocked before AIR
+ +
AIR
-
Blocked between AIR and AICAR
???????
4/25/2020 Slide #10
Complementation Analysis
4/25/2020 Cosmid pCOS106 pCOS110 pCOS115 pCOS124 Strains Complemented CE106 CE110, CE170, CE382, CE390, CE391 CE115, CE292 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 Kan R .
pur gene
• Cosmids containing pur gene mutations designated pJNXXXA.
– e.g. - CE110 pJN110A 4/25/2020 Slide #12
pCOS110 Complementation Groups
4/25/2020
pCOS110 pJN110A pJN390A pJN391A pJN170A pJN382A CE110 CE390 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 1 kb E B E E E B
4/25/2020
Completed sequence Partial sequence
Slide #14
The Purine Biosynthetic Pathway PRPP purF purD purN purLQ purM AIR TPP PRPP = 5'-Phosphoribosyl-1'-Pyrophosphate AIR = 5-aminoimidazole ribonucleotide AICAR = 5-aminoimidazole-4-carboxamide ribonucleotide TPP = Thiamine Pyrophosphate GMP
4/25/2020
AMP IMP AICAR
Slide #15
FGAR Amidotransferase
+ H N 3 CH COO CH 2 CH 2 C O NH 2 glutamine
+
O H N C N H O H O O P O O OH OH 5'-phosphoribosyl-N- formylglycinamide FGAR
4/25/2020
+ H N 3 CH O CH 2 CH 2 C O glutamate COO
+
HN H N C N H O H O O P O O OH OH 5'-phosphoribosyl-N- formylglycinamidine FGAM
Slide #16
FGAR Amidotransferase Organization
ATP-binding, cleavage FGAR enolization glutamine amide transfer Type I Single subunit 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.
4/25/2020 Slide #17
FGAR Amidotransferase Phylogenetic Analysis
proteobacteria
cyanobacteria Gram + Deinococci Thermotoga Aquifex
4/25/2020
plants Methanogens Extreme thermophiles animals fungi Type I Type 2 unknown
Slide #18
PurY as a Third Subunit
SalI EcoRI
E.coli purL
3.9 kb
SalI SalI EcoRI
Cm R
SalI EcoRI
GFP pTrp Mob pTB93F (8.0 kb) from Daniel Gage of Sharon Long’s Lab Spc R Rep
Slide #19 4/25/2020
4/25/2020
PurY as a Third Subunit
Strain Mutation Complementation by
E.coli purL
CE106 PurF::Tn5 ?
CE110 CE170 CE390 PurY::Tn5 PurL::Tn5 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.
4/25/2020 Slide #21
Lab Members
• Alumni – Diana Burley – Kevin Ferguson – Jonathan Cook – Kathy Roberts – Matthew Georgy • Current – Mark McCleland – Kim Mistiszyn – Lori Schultz – Rachel Lawton – Laura Singer – Tom Rombold Slide #22 4/25/2020