isolation of viruses infecting marine bacteria jennifer yan mentors: dr. stephen giovannoni dr. mike...

Isolation of Viruses Infecting Marine Bacteria

Jennifer Yan

Mentors:

Dr. Stephen Giovannoni

Dr. Mike Schwalbach

Giovannoni Lab

Department of Microbiology

HHMI

A Brief Introduction

• Water covers 71% of the earth’s surface

• Regulates– Weather– Climate– Atmosphere

composition

• Biodiversity

Marine Bacteria

• Production and consumption of greenhouse gases

• Biogeochemical cycling of elements

• Form base ofmarine food web

Viruses

• Infectious particles– Bacteriophages– Protein capsid, nucleic

acid, tail apparatus

• Host range– Receptor molecule

recognition– Attachment

Viral Reproduction

• Viruses are highly abundant in ocean (~107

ml-1)

• 10x average bacterial abundance (10^5-10^6 ml-1)

• Mobilize via passive diffusion, hence need an abundant host so most are believed to infect prokaryotes

• 334• 295• 337

Noble & Fuhrman 1998

Viruses in Seawater

Viruses

• Interact with bacteria• Impact ecological processes

– carbon cycling– community composition– controlling algal blooms

• Ecophysiological impacts poorly understood

• 50% of bacterial mortality caused by viruses

• Model systems needed• Virus isolates needed

– Isolate viruses for available cultures– Pelagibacter ubique

Objectives/Aims

• Survey viral and bacterial abundances off the Oregon Coast via SYBR slides & epifluorescent microscopy

Oregon Coast Survey

Results from OCS survey

Bacterial and Viral Abundances at NH5, 10 m depth

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

10/24/0612/4/071/31/073/15/075/14/076/20/077/24/078/22/07

Collection Date

Viruses per mL

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

1.60E+06

1.80E+06

2.00E+06

Bacteria per mL

Viruses

Bacteria

Mesocosm Project

Unfiltered seawater

Unfiltered seawater +

P. ubique

Mixed

viruses

Mixed

viruses

Pulsed field gel electrophoresis

Pelagibacter ubique phage isolation

PFGE of Viral DNA

Collection of Marine Viruses

Raw seawater

1.2 μm filter

0.2 μm filter

TFF

Centrifugation

Virus concentrate

Erythrobacter litoralis

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

4.00E+06

0 1 2 5 6 7 8 9 12 13 14 15

Time (days)

Cells per mL

E. litoralis

Isolation of phages

1. Pure cultures

2. High densities

3. Inoculate cultures with viruses

4. Monitor

Added

viruses

Added viruses

Bac

teria

cel

ls m

l-1

Pelagibacter ubique

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

0 5 10 15 20 25 30 35

Time (days)

Bacterial Cells per mL

Control

Experimental

Added

viruses

Results: Phage Isolates

• Erythrobacter litoralis

• Croceibacter atlanticus

• Maritimibacter alkaliphilus

• Rhodobacteriales bacterium

Acknowledgements

• Dr. Stephen Giovannoni

• Dr. Mike Schwalbach

• Giovannoni Lab

• Dr. Kevin Ahern

• HHMI

Future Work

• TEM images

• DNA sequencing

• Characterization of viruses– Virulent– Burst size– Latency period– Host range