Transcript Isolation and Physiological Characterization of a Halophilic Host

Isolation and Physiological
Characterization of a Halophilic HostPhage 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
– fCH1, haloalkaliphilic virus  IEF showed acidic capsid
proteins
Field Site: Exportadora de Sal, Guerrero Negro,
Baja California, Sur, Mexico
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: 16.2oC – 19oC
Pond #9, gypsum crystals
Bitterns pond
Pond #9 on right side
Crystallization pond
Pond #11
Crystallizer Channel
Water Sampling Protocol
•
Visited extreme hypersaline ponds:
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–
•
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
4oC
Estimate of Abundance (MPN)
MGM
DBCM
(rich medium)
(lower carbon; trace
elements; vitamins)
Crystallization
Channel 1
0.75 x 108 /ml
0.43 x 108 /ml
Crystallization
Channel 2
2.4 x 108 /ml
1.5 x 108 /ml
Isolation of Hosts
Isolation of Hosts
Viral Isolation
Crystallizer Channel 2-B
(C2- B)
Top agar overlay method:
1ml isolated cells +
1ml pre-centrifuged H20 sample +
4mls top agar
Summary of All Viral Isolates to Date
1. Crystallizer Channel 2 – high concentration
2. Pond 11 – one plaque
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
•
•
4.
Phage-host physiological experiments
Phage characterization
Visit ESSA this fall (??) and collect water for future PFGE
viral community analysis.
Acknowledgments
(aka, the logo page)
• Jesse Dillon, advisor, Cal State Long Beach
Paul Ngo, undergraduate assistant
Lamine Diallo, undergraduate assistant
• Funding: NSF Minority Postdoctoral
Fellowship
• NASA EMERG
• Government of Mexico
& ESSA
Early Microbial Ecosystems
Research Group