isolation of viruses infecting marine bacteria jennifer yan mentors: dr. stephen giovannoni dr. mike...
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![Page 1: Isolation of Viruses Infecting Marine Bacteria Jennifer Yan Mentors: Dr. Stephen Giovannoni Dr. Mike Schwalbach Giovannoni Lab Department of Microbiology](https://reader035.vdocuments.mx/reader035/viewer/2022062407/56649d815503460f94a65ab7/html5/thumbnails/1.jpg)
Isolation of Viruses Infecting Marine Bacteria
Jennifer Yan
Mentors:
Dr. Stephen Giovannoni
Dr. Mike Schwalbach
Giovannoni Lab
Department of Microbiology
HHMI
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A Brief Introduction
• Water covers 71% of the earth’s surface
• Regulates– Weather– Climate– Atmosphere
composition
• Biodiversity
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Marine Bacteria
• Production and consumption of greenhouse gases
• Biogeochemical cycling of elements
• Form base ofmarine food web
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Viruses
• Infectious particles– Bacteriophages– Protein capsid, nucleic
acid, tail apparatus
• Host range– Receptor molecule
recognition– Attachment
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Viral Reproduction
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• 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
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Viruses
• Interact with bacteria• Impact ecological processes
– carbon cycling– community composition– controlling algal blooms
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• 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
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• Survey viral and bacterial abundances off the Oregon Coast via SYBR slides & epifluorescent microscopy
Oregon Coast Survey
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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
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Mesocosm Project
Unfiltered seawater
Unfiltered seawater +
P. ubique
Mixed
viruses
Mixed
viruses
Pulsed field gel electrophoresis
Pelagibacter ubique phage isolation
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PFGE of Viral DNA
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Collection of Marine Viruses
Raw seawater
1.2 μm filter
0.2 μm filter
TFF
Centrifugation
Virus concentrate
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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
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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
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Results: Phage Isolates
• Erythrobacter litoralis
• Croceibacter atlanticus
• Maritimibacter alkaliphilus
• Rhodobacteriales bacterium
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Acknowledgements
• Dr. Stephen Giovannoni
• Dr. Mike Schwalbach
• Giovannoni Lab
• Dr. Kevin Ahern
• HHMI
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Future Work
• TEM images
• DNA sequencing
• Characterization of viruses– Virulent– Burst size– Latency period– Host range