Dual-laser flow cytometry reveals Synechococcus pigment type diversity

Synechococcus cyanobacteria exhibit a highly diverse composition of photosynthetic pigments. The fluorescence excitation ratio (Ex 495:545 ) of the blue light-absorbing photosynthetic pigment phycourobilin (PUB; λ max = 495 nm) to the green light-absorbing photosynthetic pigment phycoerythrobilin (PEB; λ max = 545 nm) differs between the pigment subtypes within Synechococcus pigment Type 3, which is predominantly found in marine waters. In the present study, we developed a method to assess the Ex 495:545 of natural Synechococcus subpopulations that were distinguished using a flow cytometer equipped with both blue (488 nm) and green (532 nm) lasers. A significantly positive linear relationship of Synechococcus strains was obtained between spectrofluorometry and dual-laser flow cytometry ( y = 0.542 x + 0.08). Using this equation, the Ex 495:545 values of natural Synechococcus subpopulations were enumerated. An Ex 495:545 below 0.60 only occurred in the coastal areas and co-occurred with other subpopulations with higher Ex 495:545 , ranging from 0.73 to 1.80. The co-occurrence of multiple Synechococcus subpopulations with different Ex 495:545 was also observed along the coastline of Japan. However, in the open ocean, only 1 subpopulation was observed, with Ex 495:545 above 1.63 at the surface and increasing by depth to 2.13. The application of this method to enumerate Ex 495:545 will advance the study of pigment type diversity and the light acclimation strategy of Synechococcus in marine environments.