The Secret Life of BacteriophagesLysogenic interactions

in soil ecosystems

Kurt WilliamsonWommack LabMarch 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

Temperate replication

Temperate replication

Environmental Signal (induction)

Temperate replication

Immunity to infections from other, similar viruses

In bacteria: lysogenic conversionDiptheria 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 plate Broth culture

Gauging Induction

40-mlLog-phase cultures1 2

T2C2T1C1

Split into duplicate 20-ml cultures

Incubate 18h, 28C, 150 rpm

Enumerate viruses & bacteria with EFM

Initial ResultsSoil pH OM %W %

sand % silt

% clay

Lysogenic fraction

Matapeake silt loam 5.9 1.7 22 9 75 16 2/5 Evesboro loamy sand 6.1 0.1 11 85 12 3 2/5 Blackbird Wetland 4.9 12.4 175 39 56 16 1/5 Piedmont Wetland 5.9 3.0 60 59 36 5 1/5

• 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 TrialsConducted by Yingbo Wang with Matapeake isolate, MP3

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

VD

C/m

l

-260.00

-210.00

-160.00

-110.00

-60.00

-10.00

40.00

90.00

140.00

1

bu

rst

size

MC MCMC30 UV MC30 UV

* *Prophage Induced Burst Size

Induction Results

Induction Results

Characterization of inducible temperate

phages

Genome Sizing

48.5

97

145.5

33.4

19.3

12.2

MP3 MP4 PW4 EV3 EV5 BW3λ M λ λM M

Genome Sizing

48.5

97

145.5

33.4

19.3

12.2

MP4λ M λ λM M

36

72

108

48

56

Genome Sizing

48.5

97

145.5

33.4

19.3

12.2

PW4λ M λ λM M

35

70

105

140

Genome Sizing

48.5

97

145.5

33.4

19.3

12.2

EV3λ M λ λM M

18

28

50

100

TEM MorphologyMP4

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 byEPA STAR Graduate Fellowship

K. Eric Wommack

Kirk CzymmekDebbie Powell

Bekki HeltonJen SchnitkerMatt SimonSharath SrinivasiahDanielle Winget

Mark Radosevich, R.S.G.

Shannon Williamson

Yingbo Wang