Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab

The goal of these projects is to quantify physiological parameters in viral infections, then map ohmic substrates to the observed physiology, leading to models or laws in virology that predict changes in virus-host dynamics. With advances in high-throughput sequencing and streamlined bioinformatics workflows, multi-ohmics analyses are readily accessible, which provides an opportunity to explain physiological phenomena as a function of specific ohmic substrates. Using experimental infections, electron microscopy, quantitative PCR next-generation sequencing, and robust bioinformatics pipelines, network analyses can identify relationships between ohmics and physiological variables which inform the development of predictive models.

The biggest challenge is generating pure single genotype virus stocks with accurate titers so that multiplicity of infection can be determined for experimental infections. Titer determination remains an issue in virology. We've developed metrics to calculate relative virulence between two viruses infecting a host or one virus infecting different hosts in single-host/single-virus infections.

We've standardized a workflow that works across virus systems. To begin, perform environmental sampling offshore along the Southern California coastline. Attach a 10-foot-long collapse-resistant rubber flex hose to the outlet of the pump.

Plug the transfer pump electrical cord into the inverter generator. Once assembled, prime the transfer pump and test to ensure the pump system is operational. Secure one end of a 25-meter-long nylon marine rope, having a diameter of 1.25 centimeters, to the eyelet of a 14-kilogram mushroom anchor.

Use duct tape to secure the rope to the blue PVC tubing every three meters. Once a steady flow of water is being transferred, discharge one to two tube volumes back into the ocean to ensure that water from the desired depth is flowing through the pump. Collect water samples in 20-liter water jugs to transport them to the field station.

Filter the water sample through a filter cup assembly, then connect the filter cup to a portable air diaphragm vacuum pump using clear vinyl tubing. Transfer the sample into the filter cup and begin filtration. When complete, transfer the filtrate into a water collection bottle and label it.

Now use a scalpel to cut out the filter from the filter cup assembly. Cut it in half and transfer into a 50-milliliter centrifuge tube. Transfer 50 milliliters of the filtered sea water into the same centrifuge tube.

To concentrate mixed samples, for each sampling depth by site, ship back the ultrafiltrate with a cell debris-laden 0.45 micrometer filter to the laboratory and centrifuge. Transfer 60 milliliters of the filtrate into a spin filter. Centrifuge it at 6, 000 G for 10 to 20 minutes to concentrate it.

Repeat centrifugation until a 1.5 to 2 milliliter concentrate of the virus or bacteriophage suspension is obtained. Transfer the resultant concentrate into a two-milliliter Eppendorf tube for further transmission electron microscopy imaging of viruses or bacteriophage from the processed environmental sample. Streak environmental samples onto prepared agar plates.

To isolate single-colony isolates of putative virus or bacteriophage hosts, use a sterile inoculation loop to transfer an isolate to a new plate. Then use the same loop to pick another colony and transfer it into a conical flask containing liquid broth. Prepare 10 seed cultures in 20-milliliter culture tubes containing five milliliters of BG 11 media.

Add five milliliters of a mix of ultrafiltrate from the sample site and BG-11. Pick a single colony isolate from a culture plate with a sterile toothpick, then inoculate each tube individually by dropping one toothpick into each tube. Once positive seed culture growth is detected, repeat streak procedures until only a single colony phenotype appears on plates.

Once cycles of liquid culture and solid media plate work result in a single cell phenotype, characterize microbial morphotypes using light microscopy. To detect virus activity, grow putative host lawns of cyanobacteria or microalgae from working stocks on BG-11 agar plates. Once individual plaques appear on plates from spotting on or swirling on virus suspension, use a sterile loop to scrape a single plaque.

Swirl the loop in a 10-milliliter seed culture of the host. To determine host range, prepare lawns of multiple putative hosts isolated from environmental samples or obtained from other laboratories or culture collections. Generate host growth curves by taking optical density at 730 nanometer readings at regular time intervals from infected and uninfected cultures.

Pure cultures of photosynthetic microorganisms displayed distinct morphologies under light microscopy. Based on morphological features and 16S rRNA sequencing followed by phylogenetic analysis, the isolate was identified as being a species of cyanobacteria from the family, Merismo PDAC. An alternative isolate showing oval-shaped green cells is suggestive of Synechococcus.

The Synechocystis like strain SWII form dense, uniform lawns on BG-11 agar, ideal for plate-based plaque assays. FSBL 14 infection caused visible plaque clearing in swirl-on lawn serial dilution plaque assays and impeded the growth of the strain SWII in liquid culture. Growth curves showed that SWII infected with concentrated phi SBL 14 suspension had a much slower increase in optical density compared to both the diluted phage suspension and the uninfected control.

Maximum specific growth rate was highest in uninfected SWII cultures. Transmission electron microscopy of infected SWII cultures revealed cytopathic effects including membrane thickening, internal granules, and membrane blabbing, which were absent in uninfected controls.