Bioluminescence in Vibrio spp.: A Mechanism for Redox Homeostasis?

Cells rely intimately on dinucleotide cofactors for metabolism. Various metabolic pathways elicited under certain environmental conditions may shift the redox balance of dinucleotide pools, necessitating mechanisms to restore balance. Bioluminescence in Vibrio species oxides NADH and NADPH, generating NAD+ and NADP+ for metabolism. This study aimed to test if bioluminescence maintains redox homeostasis under conditions of low oxygen and reductive stress. Methods I. Isolation and Identification of Bioluminescent Microbes 
 Isolate 167 (Vibrio sp.) was purified on ‘Seawater Complete’ medium (SWC) at 30 oC from an environmental sample collected from a boat launch site in Woods Hole, MA. 16s rDNA was amplified using universal PCR primers. 16s rDNA amplificons were sequenced and compared to a non-redundant nucleotide database using BLAST. II. Growth of Strains 
 Isolated strain 167 was maintained on SWC agar medium at room temperature. The Vibrio fischeri strains ES114 (WT), EVS102, and AMJ2 were obtained from Eric Stabb at the University of Georgia. V. fischeri strains were maintained on SWC agar at room temperature.
 For growth of strains, SWC liquid medium was inoculated from a single colony on agar, and grown overnight at 30 oC. Overnight liquid cultures were used to inoculate SWC medium (1:100), and the dilute cell suspension was aliquoted into multi-well plates for use in OD or luminescence measurements (96or 24-well plates). Mineral oil was applied over liquid in each well to prevent evaporation. Cell suspensions were harvested as needed from wells throughout experiments. III. Optical Density and Luminescence Measurements 
 Optical density (OD) and luminescence were measured with a Promega plate reader. OD was measured by absorbance at 600 nm, and luminescence as total light emission with 200 ms integration, or with a 495 BP filter set. Measurements were recorded for 20 hours, at 10 minute resolution. Plates were shaken for 3 seconds prior to measurements with a 2 mm shaking radius. IV. Oxygen Electrode Measurements
 24-well plates were profiled for oxygen concentration as a function of depth. Briefly, a clark-type oxygen microelectode was used to probe the depth of wells containing SWC medium (inoculated or uninoculated) in 300 μm step size for a total depth of 8000 μm. V. NAD(P)(H) Measurements
 NAD+, NADH, NADP+, and NADPH measurements were performed according to the method detailed in Kern, Price-Whelan, and Newman (Springer Science, 2014). Briefly, cells were centrifuged, lysed in 0.2 M HCl or NaOH at 50 oC for 10 minutes, quenched on ice for 5 minutes, neutralized with 0.1 M NaOH or HCl, centrifuged,