We have applied the highly sensitive fluorescent-based approach to quantify protein synthesis and individual cell growth rate in natural assemblage of associated and free heterotrophic bacteria. (This method was developed by Ty Samo, a graduate student in the laboratory who recently graduated and joined Dave Karl’s group). Bacteria are incubated with homopropargylglycine (HPG) that is a methionine analog and will be incorporated in the newly synthesized proteins. Click-IT chemistry was used to covalent bind Alexa Fluor® 488 hydrazide and HPG compounds through the reaction of alkyne and azide to form a stable triazole.
Using this method at the laser scanning confocal microscope A1R (Nikon), we have obtained a view of the activity continuum of bacteria off Scripps Pier. We found that 42% of the conjoint heterotrophic bacteria were growth-positive (positive for de novo protein synthesis) whereas 98% of the free heterotrophic bacteria were growth-positive. This result is consistent with the finding that some of the associations involve antagonistic interactions. This method also enables quantification of individual cell protein synthesis--whether the conjoint heterotrophic bacteria grew faster, on average, than the free bacteria. These data are currently being analyzed to determine growth advantage or disadvantage of bacteria associations with Synechococcus. Translating the protein synthesis rates into individual cell growth rates should enable us to determine the effect of individual microbes’ interactions on its growth rate.
Figure 3: Epifluorescence image natural marine bacteria assemblage incorporating HPG, homopropargylglycine-ALEXAFluor 488.
Bacteria-cyanobacteria associations are phylogenetically diverse