Fig. 3. Bacterial NO production by denitrification is involved in triggering algal death.

A Algal growth (bars) and bacterial growth (lines) in co-cultures with WT bacteria (green bar and line) and Δ262 bacteria cured from the native 262 kb plasmid (gray bar and black line). Each data point consists of 3 biological replicates, error bars designate ±SD. Statistical significance was calculated using a two-sample t test to compare algal cell counts in co-cultures with WT bacteria versus in co-cultures with Δ262 bacteria. Algal cell counts were significantly different on day 14 and 17 and resulted in p < 0.01. Statistical significance was calculated using a two-sample t test to compare the growth of WT bacteria versus Δ262 bacteria in co-cultures and did not result in significant differences. B Algae were co-cultured with either WT, ΔnirK or ΔnnrS→nirV bacteria. Co-culturing was conducted as described in materials and methods. Algal death was monitored manually every 6–8 h and death was defined as the complete change of the culture color from green to white. The timing of algal death was determined as follows: Co-cultures grew for 10 days and then algal death was monitored. The first culture that exhibited algal death was enumerated as 1 h, and the timing of algal death in all other co-cultures was measured from that point on (i.e. a culture that exhibited algal death 48 h after the first culture, was enumerated as 48). Each type of co-culture (with WT, ΔnirK or ΔnnrS→nirV bacteria) included 6 biological replicates. Box-plot elements are: center line—median; box limits—upper and lower quartiles; whiskers—min and max values, point—outlier. Statistical significance was calculated using a two-sample t test to compare timing of algal death between co-cultures with WT versus ΔnirK bacteria and between WT versus ΔnnrS→nirV bacteria. The difference between the timing of algal death in co-cultures with WT versus ΔnnrS→nirV bacteria was significant and resulted in p < 0.01. C Relative expression of nirK in bacterial cells from an algal-bacterial co-culture (corresponding to co-cultures in A), sampled on the indicated days. Data is relative to the expression on day 10. Each data point consists of 4 biological replicates, error bars designate ±SD. ND stands for not detected. D Fluorescence of algal cells stained with the fluorescent NO-indicator diacetate DAF-FM, incubated with live or dead bacteria for 2 h under the following conditions: control (– nitrite, light green), exposure to nitrite ( + nitrite [10 µM], dark green), or exposure to a chemical NO donor ( + NO donor, [300 µM] DEANO, black). Results represent 3 biological replicates, each containing 10,000 algal cells and 10⁷ bacterial cells when indicated. Error bars designate ±SD. Statistical significance was calculated using a two-sample t test to compare DAF-FM fluorescence between treatments. DAF-FM fluorescence between algal treatment with or without nitrite versus NO donor treatment was significant and resulted in p < 0.01. DAF-FM fluorescence of algae treated with live bacteria with nitrite versus without nitrite was significant and resulted in p < 0.01. E The effect on the death of axenic algal cultures upon adding (+) a NO donor (a single dose of 100 µM DEANO) and a NO scavenger (20 µM c-PTIO). Red checkmark indicates observed algal death. Results represent at least 3 biological replicates. ND stands for not detected.