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 of
marine food web
Viruses
• Infectious particles
– Bacteriophages
– Protein capsid, nucleic
acid, tail apparatus
• Host range
– Receptor molecule
recognition
– Attachment
Viral Reproduction
Viruses in Seawater
• Viruses are highly
abundant in ocean (~107
ml-1)
• 334
• 295
• 337
• 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
Noble & Fuhrman 1998
Viruses
• Interact with bacteria
• Impact ecological processes
– carbon cycling
– community composition
– controlling algal blooms
Objectives/Aims
• Ecophysiological impacts poorly
understood
• 50% of bacterial mortality caused by
viruses
• Model systems needed
• Virus isolates needed
– Isolate viruses for available cultures
– Pelagibacter ubique
Oregon Coast Survey
• Survey viral and bacterial
abundances off the
Oregon Coast via SYBR
slides & epifluorescent
microscopy
Results from OCS survey
2.00E+06
8.00E+07
1.80E+06
7.00E+07
1.60E+06
1.40E+06
6.00E+07
1.20E+06
5.00E+07
1.00E+06
4.00E+07
8.00E+05
3.00E+07
6.00E+05
2.00E+07
Viruses
1.00E+07
Bacteria
0.00E+00
4.00E+05
2.00E+05
0.00E+00
Collection Date
Bacteria per mL
9.00E+07
10
/2
4/
06
12
/4
/0
7
1/
31
/0
7
3/
15
/0
7
5/
14
/0
7
6/
20
/0
7
7/
24
/0
7
8/
22
/0
7
Viruses per mL
Bacterial and Viral Abundances at NH5, 10 m depth
Mesocosm Project
Unfiltered
seawater
Mixed
viruses
Pulsed field gel
electrophoresis
Unfiltered seawater
+
P. ubique
Mixed
viruses
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
Isolation of phages
Erythrobacter litoralis
4.00E+06
3.50E+06
3.00E+06
Cells per mL
1. Pure
cultures
2. High
densities
3. Inoculate
cultures
with viruses
4. Monitor
2.50E+06
Added
viruses
2.00E+06
1.50E+06
1.00E+06
5.00E+05
E. litoralis
0.00E+00
0
1
2
5
6
7
8
Time (days)
9
12
13
14
15
Pelagibacter ubique
Bacterial Cells per mL
2.50E+06
2.00E+06
Added
viruses
1.50E+06
1.00E+06
Control
5.00E+05
Experimental
0.00E+00
0
5
10
15
20
Time (days)
25
30
35
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