Characterization of non-fluorescent 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 onlyin 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 Pseudomonasfluorescens • 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 mutantschosen 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 Pyoverdine+Fe 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 # 3 4 6 7 6 5 4 3 2 1 1 2 5 7 8 mini-Tn5 Probe SphI-digest NcoI-digest Flipped compared to the gel membrane 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 gel

14. Southern analysis continued: Size markers 23,130 bp 3 4 5 6 7 8 Mutant Size of Bands 9,416 bp 3 <23130 4 15000 5 <23130 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 <564, 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