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 $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

3. Background 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

4. Background information mini-Tn5 A506 Genome mini-Tn5 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

5. Fluorescence of Pseudomonas fluorescens Pyoverdines are a class of siderophores (chelating compounds produced by organisms) A506 Fe II Fe III Fluorescence under UV is caused by a pyoverdine Siderophores are produced in iron-deficient environments, such as aerial plant surfaces receptor

6. 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

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. Pyoverdine Siderophore-mediated Iron Uptake by A506 PyoverdineReceptor FeEDDHA EDDHA Pyoverdine+Fe A506 Fe III EDDHA

9. Pvd Utilization of a Pyoverdine by Non-fluorescent Mutants A506 Pvd -

10. 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 A506 Mutant 8 Four non-fluorescent mutants

11. Next 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

12. 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 NcoISphI 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

13. Southern analysis continued: mini-Tn5 Probe Hybridization After probe is applied, membrane is washed in a visualization solution Flipped compared to the gel gel membrane Mutant # 12 3 5 6 87 4 235674 NcoI-digest 8112345678 SphI-digest 1234567

14. Southern analysis continued: Size markers 23,130 bp 9,416 bp 6,557 bp 4,361 bp 2,322 bp 2,027 bp 876543 MutantSize of Bands 3 <23130 4 15000 5 <23130 6 7200 7 6900, 4300 8 -

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 iii.Run PCR rxn. ii. Ligate digested genomic DNA into circular DNA i.Cut genomic DNA with restriction enzyme Steps: mini Why is it called inverse-PCR?

17. 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.

18. Progress 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

19. 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

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 TempleMeg Roche Larsen Brenda SchafferAmy Davis Marcella HenkelsAndy Mumford