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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