Combining fluorescence microscopy and NanoSIMS.

<p>A The cells were grown in well-mixed liquid cultures–batch or chemostats, fixed, and then deposited to the filters for NanoSIMS analysis. Therefore, cells that are close to each other on the filter were not close during their growth. For individual cells, we measured expression of ParaE-<i>gfp</i>, expression of PptsG-<i>mCherry</i>, ¹³C atom fraction and ²H atom fraction in the same area of interest. We analyzed only those cells that could be unambiguously defined and linked between raw NanoSIMS and fluorescence microscopy images (colored cells; other cells are depicted in grey). B Single-cell measurements of three independent replicate cultures (depicted in three different colors) grown in carbon-limited chemostats (10 μM of each labeled sugar) and carbon-excess batch cultures (1.5 mM of each labeled sugar) were plotted. There was no significant correlation between ²H excess atom fraction <i>X</i> ^<i>E</i>(²H)_cell and PptsG-<i>mCherry</i> expression regarding glucose utilization (Spearman's rho = 0.120, p = 0.373, N = 57 cells in chemostats; rho = 0.063, p = 0.579, N = 81 cells in batch), nor between ¹³C excess atom fraction <i>X</i>^<i>E</i> (¹³C)_cell and ParaE-<i>gfp</i> expression regarding arabinose utilization (rho = 0.031, p = 0.821 in chemostats; rho = -0.197, p = 0.078 in batch) in neither of the two tested conditions. C We determined transcriptional specialization (PptsG-<i>mCherry</i> signal relative to ParaE-<i>gfp</i> signal) and sugar specialization (²H excess atom fraction relative to ¹³C excess atom fraction) for cells grown in carbon-limited chemostats, as defined in the Methods section ‘Analysis of filters with fluorescence microscopy and NanoSIMS’. Cells that have higher PptsG-<i>mCherry</i> signal relative to the ParaE-<i>gfp</i> signal also have higher ²H excess atom fraction relative to the ¹³C excess atom fraction, and <i>vice versa</i>. In other words, cells that specialize more on the <i>ptsG</i> transcription tend to have a stronger specialization on glucose assimilation. This observation is based on a positive parametric correlation between transcriptional specialization and sugar specialization (Pearson's r = 0.304, p = 0.021, N = 57 cells from 3 replicates depicted in different colors; non-parametric correlation analysis is not significant).