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 are highly abundant in ocean (~10 7 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 Noble & Fuhrman 1998 Viruses in Seawater

Viruses Interact with bacteria Impact ecological processes –carbon cycling –community composition –controlling algal blooms

Jenn I think you need to list your main objectives this summer along with the broad question etc.. (e.g. why did you do this.) Broad question: Given that viruses can account for 50% of bacterial mortality, people (including you) interested in understanding how viruses impact bacterial population structure and physiology – to do this we need more model systems, hence more isolates should be obtained Our goal was to isolate novel marine viruses using unique bacterial hosts available in the Giovannoni lab culture collection Characterize bacterial/viral populations off the Oregon coast Specifically interested in isolating viruses for Pelagibacter ubique (SAR11) – the worlds most abundant bacteria 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

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

Isolation of phages 1.Pure cultures 2.High densities 3.Inoculate cultures with viruses 4.Monitor Added viruses

Added viruses Bacteria cells ml-1 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