1. The Secret Life of Bacteriophages Lysogenic interactions in soil ecosystems Kurt Williamson Wommack Lab March 9, 2006

2. Introduction - terms Phage – bacteriophage, a virus that infects bacteria. Temperate phage – virus able to integrate into its host’s chromosome. Prophage –integrated virus genome. Lysogen – a bacterium containing an integrated temperate phage.

3. Temperate replication

5. Environmental Signal (induction)

6. Temperate replication

7. Immunity to infections from other, similar viruses In bacteria: lysogenic conversion Diptheria toxin (Barksdale and Pappenheimer, 1954) Cholera toxin (Waldor and Mekalanos, 1996) Less dramatic examples: Prophage down-regulates host metabolism (Chen et al., 2005) … may be significant evolutionary repercussions. Impact of Temperate Infections

8. How prevalent are lysogenic interactions in soils? Culture-dependent approach

9. Sampling Locations White Clay Creek Preserve Ag Experimental Stn., Newark Blackbird Forest Ag Experimental Stn., Georgetown

10. Approach Isolate bacteria from soils; grown on ¼ TSA/TSB. Spread plate Streak plateBroth culture

11. Gauging Induction 40-ml Log-phase cultures 12 T2C2T1C1 Split into duplicate 20-ml cultures Incubate 18h, 28C, 150 rpm Enumerate viruses & bacteria with EFM

12. Initial Results Agricultural soils: 2 of 5 isolates harbored inducible temperate phages. Wetland soils: 1 of 5 isolates contained inducible phages. Overall: 6 of 20 isolates tested (30%) contained inducible phages.

13. Induction Trials Conducted by Yingbo Wang with Matapeake isolate, MP3 MC MC30UVMC30UV * * Prophage InducedBurst Size

14. Induction Results

16. Characterization of inducible temperate phages

17. Genome Sizing 48.5 97 145.5 33.4 19.3 12.2 MP3MP4 PW4 EV3EV5BW3 λ M λλ M M

18. Genome Sizing 48.5 97 145.5 33.4 19.3 12.2 MP4 λ M λλ M M 36 72 108 48 56

19. Genome Sizing 48.5 97 145.5 33.4 19.3 12.2 PW4 λ M λλ M M 35 70 105 140

20. Genome Sizing 48.5 97 145.5 33.4 19.3 12.2 EV3 λ M λλ M M 18 28 50 100

21. TEM Morphology MP4 BW3 MP4MP3 EV3 PW4EV5

22. Conclusions Lysogens/inducible temperate phages found in all 4 soils. Six of 20 soil isolates contained inducible phages. Treatment with mitomycin C resulted in higher induction responses than UV exposure. Treatment with mitC for 30 resulted in significantly higher induction response and burst sizes for some (but not all) isolates. Induction response and burst size depends on specific induction conditions…

23. Conclusions Five of six lysogens produced siphophage particles with genome sizes similar to lambda – these lysogens are Bacillus species. One of the six lysogens produced small numbers of myophage particles with very small genome (12 KB) – host is Ochrobactrum spp. Future work: Community inductions from same 4 soils.

24. Thanks Funded by EPA STAR Graduate Fellowship K. Eric Wommack Kirk Czymmek Debbie Powell Bekki Helton Jen Schnitker Matt Simon Sharath Srinivasiah Danielle Winget Mark Radosevich, R.S.G. Shannon Williamson Yingbo Wang