Characterization of nonfluorescent mutants of Pseudomonas fluorescens A506

1
Characterization of non-fluorescent mutants of
Pseudomonas fluorescens A506

• Student researcher
• Kevin Hockett
• Mentor
• Dr. Virginia Stockwell
• USDA ARS
• Loper Lab

2
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

3
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 .

4
Background information

• A graduate student in the lab created a
  collection of twenty-three mini-Tn5 km mutants of
  A506 that are non-fluorescent

parental strain A506
Tn5 non-fluorescent mutant number 8
mini-Tn5
mini-Tn5
A506 Genome
A506 Genome
A506 Genome
mini-Tn5
mini-Tn5
5
Fluorescence of Pseudomonas fluorescens

• Fluorescence under UV is caused by a pyoverdine

• Pyoverdines are a class of siderophores
  (chelating compounds
• produced by organisms)

receptor
A506

• Siderophores are produced in iron-deficient
• environments, such as aerial plant surfaces

Fe II
Fe III
6
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
7
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
8
Siderophore-mediated Iron Uptake by A506
EDDHA
FeEDDHA
PyoverdineFe
Pyoverdine
EDDHA
A506
Pyoverdine Receptor
Fe III
9
Utilization of a Pyoverdine by Non-fluorescent
Mutants
A506
Pvd
Pvd-
10
A506
Four non-fluorescent mutants
Mutant 8

• Conclusions
• 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

11
Next step

• Investigate the gene that has been disrupted

Putative regulatory gene disrupted by mini-Tn5
insertion
mini-Tn5

X
Mutant A506 Genome
Antibiotic
Pyoverdine
How to achieve?
12
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
Second separate digested DNA on gel based on
size
mini-Tn5
NcoI
SphI
Not good representation
Digested Genomic DNA
Third Blot the gel (transfer DNA from gel to a
nylon membrane)
13
Southern analysis continued
After probe is applied, membrane is washed in a
visualization solution
Hybridization
Mutant
1
2
3
5
6
8
7
4
1
2
3
4
5
6
7
mini-Tn5
Probe
SphI-digest
NcoI-digest
Flipped compared to the gel
membrane
2
3
5
6
7
4
8
1
1
2
3
4
5
6
7
8
gel
14
Southern analysis continued
Size markers
23,130 bp
8
7
6
5
4
3
Mutant
Size of Bands
9,416 bp
3 lt23130 4 15000 5
lt23130 6 7200 7
6900, 4300 8 -
6,557 bp
4,361 bp
2,322 bp
2,027 bp
15
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
• Number
  Representative Enzymes
• Mutant of insertions NcoI
  SphI PstI
• 8 1 4150,
  9144 2690, 6400 810, 1720, 4512
• 7 2 1200, 5500 1768,
  6860,
• 9039, 11094
• 6 1 lt564, 8800 5084,
  7136

16
Inverse PCR
Inverse PCR a method to amplify DNA adjacent to
mini-Tn5 for sequencing
Steps

• Cut genomic DNA with restriction enzyme

mini

• Ligate digested genomic DNA into circular DNA

mini
mini

• Run PCR rxn.

Why is it called inverse-PCR?
17
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.
18
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

19
Conclusions

• 22 of 23 non-fluorescent mutants of A506 were
  unable to grow on media amended with EDDHA
• One mutant grew on EDDHA and cross-fed all other
  mutants
• All non-fluorescent mutants could be cross-fed on
  iron-depleted media by the parental strain A506.
• Of eight mutants evaluated with Southern
  analysis, seven had a single insertion of Tn5
• 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

20
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