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

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

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.

Temperate replication

Environmental Signal (induction)

Temperate replication

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

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

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

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

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

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.

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

Induction Results

Characterization of inducible temperate phages

Genome Sizing MP3MP4 PW4 EV3EV5BW3 λ M λλ M M

Genome Sizing MP4 λ M λλ M M

Genome Sizing PW4 λ M λλ M M

Genome Sizing EV3 λ M λλ M M

TEM Morphology MP4 BW3 MP4MP3 EV3 PW4EV5

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…

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.

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

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