Measuring the Number of LuxR Proteins in a Single Cell of V. harveyi

We have determined the number N of LuxR proteins in a single cell of the bacterium V. harveyi by measuring the distribution functions of cell volumes and protein-fluorescence intensities during cell division. In quorum sensing, the LuxR protein population, which regulates many (∼70) genes, is sensitive to the concentration of auto-inducer molecules (AIs). We utilized a strain that is incapable of producing AIs. The LuxR proteins are tagged by a red fluorescent protein (mCherry). In the absence of AIs, the cells maintain a baseline residual concentration of LuxR that is remarkably constant over 8-10 cycles of cell division. We recorded the growth of a single cell into a large colony by imaging both phase contrast and mCherry intensity every 2 minutes in a 6-hour movie. The phase-contrast image was used to measure the volume of each cell, while the mCherry intensity monitored the LuxR population. At each cell-division event, we determined the fractional partitioning of the cell volume and the LuxR population. From the large number of cell-division events (∼300), we obtained the normalized distributions of both the volumes and the mCherry intensities. Our procedure allows an accurate measurement of the width of the volume distribution (σ = 0.031± 0.003). Significantly, the width of the LuxR distribution was observed to be much broader, presumably because of small-number fluctuations. By deconvoluting the Gaussian distributions, we find that the average LuxR copy number N equals 140 ± 10 just before cell division. Repeating the experiment at successively higher levels of applied AI concentration, we confirmed that when N is 10 times larger, the 2 distributions converge to the same width σ. This technique may be applied quite generally to other systems.