Abstract:
Bioorthoganol Non-Canonical Amino Acid Tagging (BONCAT) was used in combination with fluorescence activated cell-sorting (FACS) to assess the impacts of elevated hydrostatic pressure on the protein activity of hydrocarbon degrading bacteria. Three study bacteria strains were used - Halomonas titanicae strain (Bead 10BA); Alcanivorax xenomutans strain (Bead 18); and Shewanella indica strain (Bead 36). Each strain was grown up to mid-log phase in ONR7a minimal medium supplemented with 50 mM hexadecane for each growth condition (aerobic flask, 0.1 MPa, 10 MPa, 25 MPa. BONCAT labeling using HPG was performed using the protocol outlined in Hatzenpichler et al. 2016, PNAS 113(28). Samples were then flow sorted using a ZE5 Cell Analyzer (BioRad) equipped with small-particle detection module. The DNA stain used was Hoechst 33342, and the BONCAT stain was an Alexa Flour 488 azide dye. Negative controls included PBS storage buffer and cultures not labeled with HPG, to define the level of nonspecific BONCAT stain fluorescence. The fraction of BONCAT positive cells was determined over time for the 0.1 MPa, 10 MPa, and 25 MPa incubations.
Suggested Citation:
Xu, Tianhan, Kelli K. Mullane, and Douglas H. Bartlett. 2020. Impacts of pressure on the activity of hydrocarbon degrading three strains of bacteria. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/GP2SFWZQ
Data Parameters and Units:
The headers are: Time (hours), Fraction of Active Cells, pressure (MPa), and Standard Deviation
Methods:
BONCAT was performed following the established protocol of Hatzenpichler et al. 2016 with following modifications. The cultures were initially set up in 10ml sealed serum glass vials for three strains under atmosphere pressure and high pressures, respectively, to promote growth. When they reached the mid-log phase, as determined by prior protein assay-based growth curve analysis, HPG was added. Specifically, 1 μl HPG (50mM) were added into the 10 ml enriched cultures and inverted gently to mix (final concentration of HPG is 5μM). The vials were then labeled, and re-incubated at the desired pressures. Samples (10 ml) were withdrawn as a function of time, fixed with 8.8 μl paraformaldehyde to a final concentration of 3% (15 minutes at room temperature, in the dark). Then the cells were pelleted via centrifugation (16,900 g or max, for 5 mins at RT), supernatants removed, and the cell pellets resuspend in 100 μl PBS. This cell suspension was pelleted as above to withdraw any remaining paraformaldehyde, resuspended in 100μl 1:1 PBS: EtOH, and stored at -20 degrees Celsius until further treatment. For click chemistry, the samples were thawed and pelleted as above, followed by removal of the supernatant and resuspension in 100 μL PBS. The “click cocktail” was always freshly mixed according to the instructions of the click kit (Click-iT HPG Alexa Flour Protein Synthesis Assay Kits, Life Technologies). One hundred microliters of this cocktail solution were added into each sample and incubated in the dark at room temperature for 30 minutes. After click chemistry, the cells were washed with 100 μl rinse buffer and 3% BSA respectively, via centrifugation (16,900 g or max, for 5mins at RT). Finally, the cells were resuspended in 500 μl PBS, and DNA stain was added. Hoechst 33342 was used in this study, diluted to final concentration of 10 μg/ml. The stained cells were subsequently examined with flow cytometry.
Samples were run on a ZE5 Cell Analyzer (BioRad) equipped with the small-particle detection module. DNA stain Hoechst 33342 (excitation = 361 nm, emission = 497 nm) was excited off the 355nm laser (50mW) and fluorescence was collected through a 447/60 nm band-pass filter, while the Alexa Flour 488 azide dye (excitation = 490 nm/ emission = 525 nm) was excited off the 488 nm laser (100mW) and fluorescence collected through a 523/30 nm band-pass filter. Sample delivery was by a calibrated peristaltic pump allowing for precise measurement of absolute counts. The PBS buffer used for the final sample resuspension was used as a negative control to exclude background particles. The first gate was drawn on the Hoechst positive (Hoechst 33342+) particles, under the assumption that this would capture the cells. Hoechst 33342+ events accounted for >95 % of the events depending on the samples. The BONCAT positive (BONCAT +) and BONCAT negative (BONCAT -) where further gated as a subfraction of the Hoechst 33342+ cells based on the Alexa Fluor 488 azide dye fluorescence. The samples that were not incubated with HPG were used as negative controls to define the level of nonspecific BONCAT stain fluorescence, the BONCAT- gate was drawn under that line and BONCAT + gate was such that <1% of negative control cells were in it. The fraction of BONCAT + cells was determined for a time course for both 0.1MPa, 10 MPa and 25 MPa. Flow cytometry data were analyzed using the program FlowJo (FlowJo LLC, BD).
Provenance and Historical References:
Hatzenpichler, Roland, Stephanie A. Connon, Danielle Goudeau, Rex R. Malmstrom, Tanja Woyke, Victoria J. Orphan. 2016. Visualizing in situ translational activity for identifying and sorting slow-growing archaeal-bacterial consortia. Proceedings of the National Academy of Sciences of the United States of America 113(28): E4069-E4078.
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