Characterization of non-fluorescent Characterization of non-fluorescent mutants of mutants of Pseudomonas fluorescensPseudomonas fluorescens

A506A506Student researcher: Student researcher: Kevin HockettKevin Hockett

Mentor: Mentor: Dr. Virginia StockwellDr. Virginia Stockwell USDA ARSUSDA ARSLoper LabLoper Lab

Why is the bacterium A506 important?

•Commercial biocontrol agent for fire blight

• $68,000,000 in damage in Oregon and Washington due to fire blight in 1998

•Fire blight is a bacterial disease of pear and apple trees caused by Erwinia amylovora

Background informationBackground information

•A506 produces an antibiotic toxic to E. amylovora only in media containing excess iron

•Received two mutants of A506 always make the antibiotic in culture (iron is no longer required). These mutants are non-fluorescent .

•In several experiments in orchards, adding iron to A506 improved control of fire blight

Background informationBackground information

mini-T

n5

mini-Tn5

mini-Tn5

A506 Genome A506 Genome A506 Genome

mini-T

n5

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

parental strainA506

Tn5 non-fluorescentmutant number 8

Fluorescence of Fluorescence of PseudomonasPseudomonas fluorescensfluorescens

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

A506

Fe II

N NH

OH

O

O

NHOH

CH3

O

NH

ONH

O

OH

OH

O

CH3NH

O

NH2

NH

O

NN

OH

OH

NH

O

NH2

O

CH3

Fe III

•Fluorescence under UV is caused by a pyoverdine

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

receptor

A link between pyoverdine and antibiosis?A link between pyoverdine and antibiosis?

•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

•Two non-fluorescent mutants of A506 do not require iron to make the antibiotic in culture (from California)

•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 Investigating phenotypes of non-fluorescent mutants of A506mutants 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

PyoverdinePyoverdine

Siderophore-mediated Iron Uptake by Siderophore-mediated Iron Uptake by A506A506

PyoverdinePyoverdineReceptorReceptor

FeEDDHA

EDDHA

Pyoverdine+FePyoverdine+Fe

A506

Fe III

EDDHA

PvdPvd

Utilization of a Pyoverdine by Non-fluorescent Mutants

A506

Pvd-

ConclusionsConclusions•No receptor/uptake mutants •Mutant 8 produced a compound that cross-feed other mutants, though not a pyoverdine8 was a mutant that produced the antibiotic irrespective of iron

A506 Mutant 8 Four non-fluorescent mutants

Next stepNext step

•Investigate the gene that has been disrupted

mini-Tn5

Putative regulatory gene disrupted by mini-Tn5 insertion

How to achieve?

Mutant A506 Genome

Pyoverdine

Antibiotic

+

X

mini-Tn5

First: digest genomic DNA of mutants with various restriction enzymes

A506 Mutants :NcoI,SphI, BglI-Single cut XbaI,MluI,SpeI-No cuts

*Not good representation

Digested Genomic DNA

NcoI SphI

Second: separate digested DNA on gel based on size

Southern Analysis: Used to estimate the number of insertions and the uniqueness of their location

Third: Blot the gel (transfer DNA from gel to a nylon membrane)

Steps

Southern analysis continued:Southern analysis continued:

min

i -Tn5

Pro

be

Hybridization After probe is applied, membrane is washed ina visualization solution

Flipped comparedto the gel

gel

membrane

Mutant #

1 2 3 5 6 874

23567 4

NcoI-digest

8 1 1 2 3 4 5 6 7 8

SphI-digest

1234567

Southern analysis continued:Southern analysis continued:Size markers

23,130 bp

9,416 bp

6,557 bp

4,361 bp

2,322 bp

2,027 bp

876543Mutant Size of Bands

3 <231304 150005 <23130 6 72007 6900, 43008 -

Interpretation from Southern BlottingInterpretation from Southern Blotting

Of the 8 mutants:7 single insertions, 1 double insertionAll band patterns were unique- no insertions were in the exact

same spotwith in the genome

Number Representative EnzymesMutant: of insertions NcoI SphI

PstI

8 1 4150, 9144 2690, 6400 810, 1720, 4512

7 2 1200, 5500 1768, 6860, 9039, 11094

6 1 <564, 8800 5084, 7136

Inverse PCRInverse PCRInverse PCR: a method to amplify DNA adjacent to mini-Tn5 for sequencing

iii. Run PCR rxn.

ii. Ligate digested genomic DNA into circular DNA

i. Cut genomic DNA with restriction enzyme

Steps:

mini

mini mini

Why is it called inverse-PCR?

Inverse PCR continued:Inverse PCR continued:

Normal PCR:

Forward Primer

Reverse Primer

Inverse PCR:mini

End Primer

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 mutantsProgress in inverse PCR for non-fluorescent mutants

•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

Found

Conclusions:Conclusions:1.1. 22 of 23 non-fluorescent mutants of A506 were unable to grow on 22 of 23 non-fluorescent mutants of A506 were unable to grow on

media amended with EDDHAmedia amended with EDDHA

2.2. One mutant grew on EDDHA and cross-fed all other mutantsOne mutant grew on EDDHA and cross-fed all other mutants

3.3. All non-fluorescent mutants could be cross-fed on iron-depleted All non-fluorescent mutants could be cross-fed on iron-depleted media by the parental strain A506.media by the parental strain A506.

4.4. Of eight mutants evaluated with Southern analysis, seven had a Of eight mutants evaluated with Southern analysis, seven had a single insertion of Tn5single insertion of Tn5

5.5. Of 8 mutants evaluated with Southern analysis, each yielded a Of 8 mutants evaluated with Southern analysis, each yielded a distinct band pattern with several restriction enzymes. Each distinct band pattern with several restriction enzymes. Each mutant may have an unique insertion.mutant may have an unique insertion.

•Next step is to amplify fragments containing insert so flanking DNA can be sequenced

AcknowledgementsAcknowledgements

Howard Hughes Medical Institute Summer Fellowship ProgramDr. Kevin Ahern

USDA-Western Regional Integrated Pest Management Program

OSU Dept. of Botany and Plant PathologyDr. Virginia Stockwell

USDA/ARS Horticulture Crops Research LaboratoryDr. Joyce Loper

Todd Temple Meg Roche LarsenBrenda Schaffer Amy DavisMarcella Henkels Andy Mumford