Transcript Characterization of non-fluorescent mutants of Pseudomonas fluorescens A506 Student researcher: Kevin Hockett Mentor: Dr. Virginia Stockwell USDA ARS Loper Lab.

Characterization of non-fluorescent mutants
of Pseudomonas fluorescens A506
Student researcher:
Kevin Hockett
Mentor:
Dr. Virginia Stockwell
USDA ARS
Loper Lab
Why is the bacterium A506 important?
•Commercial biocontrol agent for fire
blight
•Fire blight is a bacterial disease of pear
and apple trees caused by Erwinia
amylovora
• $68,000,000 in damage in Oregon and Washington due to fire blight in 1998
Background information
•A506 produces an antibiotic toxic to E. amylovora only in media
containing excess iron
•In several experiments in orchards, adding iron to A506 improved
control of fire blight
•Received two mutants of A506 always make the antibiotic in culture
(iron is no longer required). These mutants are non-fluorescent .
Background information
•A graduate student in the lab created a collection of
twenty-three mini-Tn5 km mutants of A506 that are nonfluorescent
Tn5 non-fluorescent
mutant number 8
parental strain
A506
mini-Tn5
A506 Genome
mini-Tn5
A506 Genome
A506 Genome
Fluorescence of Pseudomonas fluorescens
•Fluorescence under UV is caused by a pyoverdine
•Pyoverdines are a class of siderophores (chelating compounds
produced by organisms)
•Siderophores are produced in iron-deficient
environments, such as aerial plant surfaces
receptor
A506
Fe II
O
O
NH2
N
H
N
OH
HO
Fe III
N
H
N
N
CH3
O
OH
OH
O
H
N
N
H
H2N
O
NH
O
HO
HO
O
H3C
NH
O
O
N
H
CH3
O
A link between pyoverdine and antibiosis?
•Two non-fluorescent mutants of A506 do not require iron to make the antibiotic
in culture (from California)
•Of 23 non-fluorescent, mini-Tn5 mutants:
11 no longer required iron for antibiosis
12 still required iron for antibiosis
A subset of 8 mutants chosen for further evaluation based on phenotype
•Is there a relationship between antibiosis and pyoverdine production in A506?
•Which gene(s) were affected by Tn5 insertion?
•Do all mutants of the same phenotype have similar mutations
or are all different?
•Single, double or triple insertion?
Hypothesis: At least one mutant that does not require iron for antibiosis
contains a single insertion in a regulatory gene
Investigating phenotypes of non-fluorescent mutants of
A506
Cross-feeding assay: Determine if the non-fluorescent
mutants can utilize the iron bound to the pyoverdine of A506
in iron-limited media
Siderophore-mediated Iron Uptake by
A506
EDDHA
FeEDDHA
Pyoverdine+Fe
Pyoverdine
Pyoverdine
Receptor
EDDHA
A506
Fe III
Utilization of a Pyoverdine by Non-fluorescent Mutants
A506
Pvd
Pvd-
A506
Conclusions
Mutant 8
Four non-fluorescent
mutants
•No receptor/uptake mutants
•Mutant 8 produced a compound that cross-feed other mutants,
though not a pyoverdine
8 was a mutant that produced the antibiotic irrespective of iron
Next step
•Investigate the gene that has been disrupted
Putative regulatory gene disrupted by mini-Tn5 insertion
mini-Tn5
X
Mutant A506 Genome
Pyoverdine
How to achieve?
+
Antibiotic
Southern Analysis: Used to estimate the number of
insertions and the uniqueness of their location
Steps
First: digest genomic DNA of mutants with various restriction enzymes
A506 Mutants :
NcoI,SphI, BglI-Single cut
XbaI,MluI,SpeI-No cuts
mini-Tn5
Second: separate digested
DNA on gel based on size
NcoI
SphI
*Not good representation
Digested Genomic DNA
Third: Blot the gel (transfer DNA from gel to a nylon membrane)
Southern analysis continued:
Hybridization
After probe is applied, membrane is washed in
a visualization solution
Mutant #
7 6 5 4 3 21
SphI-digest
1 2 34 5 6 7 8
NcoI-digest
membrane
8 7 654 321
12 345 6 7 8
gel
Flipped compared
to the gel
Southern analysis continued:
Mutant
3
4
5
6
7
8
Size of Bands
<23130
15000
<23130
7200
6900, 4300
-
3
4
5
6
7
Size markers
8
23,130 bp
9,416 bp
6,557 bp
4,361 bp
2,322 bp
2,027 bp
Interpretation from Southern Blotting
Of the 8 mutants:
7 single insertions, 1 double insertion
All band patterns were unique- no insertions were in the exact same spot
with in the genome
Mutant:
Number
of insertions
NcoI
Representative Enzymes
SphI
8
1
4150, 9144
2690, 6400
7
2
1200, 5500
1768, 6860,
9039, 11094
6
1
<564, 8800
5084, 7136
PstI
810, 1720, 4512
Inverse PCR
Inverse PCR: a method to amplify DNA adjacent to mini-Tn5 for sequencing
Steps:
i.
Cut genomic DNA with restriction enzyme
mini
ii.
Ligate digested genomic DNA into circular DNA
mini
iii. Run PCR rxn.
Why is it called inverse-PCR?
mini
Inverse PCR continued:
Forward Primer
Normal PCR:
Reverse Primer
End Primer
Inverse PCR:
mini
Rev. Primer
Run amplified DNA on a gel, extract, and send DNA for sequencing.
Perform a BLAST search on sequence with GenBank to help
determine identity of the disrupted gene.
Progress in inverse PCR for non-fluorescent mutants
Found
•NcoI, PstI, and SphI are
good restriction enzymes for
inverse PCR for these mutants
•Primers have been developed
and obtained for inverse PCR
from the mini-Tn5
Conclusions:
1. 22 of 23 non-fluorescent mutants of A506 were unable to grow on
media amended with EDDHA
2. One mutant grew on EDDHA and cross-fed all other mutants
3. All non-fluorescent mutants could be cross-fed on iron-depleted
media by the parental strain A506.
4. Of eight mutants evaluated with Southern analysis, seven had a
single insertion of Tn5
5. Of 8 mutants evaluated with Southern analysis, each yielded a
distinct band pattern with several restriction enzymes. Each
mutant may have an unique insertion.
•Next step is to amplify fragments containing insert so flanking DNA can be sequenced
Acknowledgements
Howard Hughes Medical Institute Summer Fellowship Program
Dr. Kevin Ahern
USDA-Western Regional Integrated Pest Management Program
OSU Dept. of Botany and Plant Pathology
Dr. Virginia Stockwell
USDA/ARS Horticulture Crops Research Laboratory
Dr. Joyce Loper
Todd Temple
Meg Roche Larsen
Brenda Schaffer
Amy Davis
Marcella Henkels
Andy Mumford