Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

In most aquatic bacterial communities, it is possible to discriminate bacterial cells with a high nucleic acid content (HNA) from those with a low nucleic acid content (LNA) by flow cytometry. The distribution of leucine incorporation rate per cell (specific activity) within the fraction of bacteria with a high apparent nucleic acid content was investigated in coastal seawater using flow cytometry and cell sorting techniques. Bacterial cells from a natural seawater sample were first labeled with tritiated leucine, stained with a fluorescent nucleic acid stain and then sorted based on their fluorescence and scatter signals, assuming that fluorescence was proportional to the cellular nucleic acid content and scatter signals to biovolume. Our results clearly demonstrated that specific activity was heterogeneously distributed within the group of HNA cells and increased with both scatter and fluorescence signals. This shows that important differences in terms of cell-specific rates of activity exist within the HNA group and that specific activity is positively correlated with both the nucleic acid content of cells and their biovolume. We hypothesized that this heterogeneity may be partly due to the structure of bacterial communities. Therefore, further investigations were made by analyzing the nucleic acid content and scatter properties of a set of 10 species isolated from the same coastal area. Our results confirm that both fluorescence and scatter values vary greatly between species at a given growth stage and between growth stages for a given species. Variations reported at different growth stages suggest that both parameters are sensitive to the metabolic activity of individual cells and confirm the positive relationship reported in this study between scatter and fluorescence parameters and specific activity within the HNA cellular fraction of natural communities. Interestingly, it was shown that HNA and LNA cells were present in late stationary phase cultures. This suggests that LNA cells found in natural communities may be dead or dying cells.