Isolation and Physiological Characterization of a Halophilic Host- Phage System from Solar Salterns in Baja California, Mexico or Extreme Halophiles: Host-Virus Dynamics

Hypersaline Environments Hypersaline >100 g/liter of total salt; extreme hypersaline > 250 g/liter (up to saturation level of salt) Thalassohaline or athalassohaline (marine composition vs. non-marine composition) Great Salt Lake, Dead Sea, solar salterns (ESSA), hypersaline alkaline lakes (East Africa, China) Abundant microbial life: –Archaeal - require very high [salt] to survive –Bacterial - tolerate wide range of salinities (low to high) –Eucaryal - (Dunaliella: red & green; Picocystis; fungi)

Questions about Halophilic Viruses What is the diversity of hosts and their phages? What is ecological range of viruses? Host specificities? How do halophilic phages affect hosts? –Bacterial community structure (host mortality) –Gene transfer Are there specific adaptations in halophage to survive in extreme hypersaline environments? –Hosts utilize more acidic amino acid residues and more salt bridges in proteins –  CH1, haloalkaliphilic virus  IEF showed acidic capsid proteins

Field Site: Exportadora de Sal, Guerrero Negro, Baja California, Sur, Mexico

Guerrero Negro Pond #9…?

Study Site: Exportadora de Sal (ESSA), Baja California, Sur, Mexico Solar saltern located in Guerrero Negro Lagoon Salinity from seawater to saturation (~48 g/l to >300 g/l) Water temperature: 16.2 o C – 19 o C Pond # 9, gypsum crystals Pond # 9 on right side Pond #11 Crystallizer Channel Bitterns pond Crystallization pond

Water Sampling Protocol Visited extreme hypersaline ponds: –Pond # 9 = 156 g/l (15.6%)  lowest salinity pond visited –Ponds # 11 & # 12 = >280 g/l (>28%) –Crystallizer Channels 1 & 2 Collected 500 mls water samples for inoculation/isolation and molecular purposes; stored at 4 o C

Estimate of Abundance (MPN) MGM (rich medium) DBCM (lower carbon; trace elements; vitamins) Crystallization Channel x 10 8 /ml0.43 x 10 8 /ml Crystallization Channel x 10 8 /ml1.5 x 10 8 /ml

Isolation of Hosts

Viral Isolation Top agar overlay method: 1ml isolated cells + 1ml pre-centrifuged H 2 0 sample + 4mls top agar Crystallizer Channel 2-B (C2- B)

Summary of All Viral Isolates to Date 1. 1.Crystallizer Channel 2 – high concentration 2. 2.Pond 11 – one plaque 3. 3.Pond 12 – one plaque

Next Steps 1.Molecular/phylogenetic analysis of initial water samples and of isolates; 2.Screen for more lytic phages and induce for temperate viruses (antibiotic treatment, UV irradiation, salinity shock); 3.Purify and grow up current viral isolates Phage-host physiological experiments Phage characterization 4.Visit ESSA this fall (??) and collect water for future PFGE viral community analysis.

Funding: NSF Minority Postdoctoral Fellowship Acknowledgments (aka, the logo page) Jesse Dillon, advisor, Cal State Long Beach Paul Ngo, undergraduate assistant Lamine Diallo, undergraduate assistant NASA EMERG Government of Mexico & ESSA Early Microbial Ecosystems Research Group

Acknowledgements Early Microbial Ecosystems Research Group ESSA & Mexican Gov’t.