Use of flow cytometry for efficient isolation of cyanobacterial mutants deficient in modulation of pigment level.

The modulation of pigment level is an essential adaptation response that allows photosynthetic organisms to adjust light harvesting to ambient conditions and to avoid oxidative damage caused by excess absorbed light (1,2). Cyanobacteria possess a macromolecu-lar pigment complex termed phyco-bilisome, which harvests light energy and transfers it to the photosynthetic reaction center (3). This ultra-structure, which may comprise up to 50% of the soluble cellular protein, contains various apoprotein subunits, several different chromophors, as well as nonpig-mented linkers required for assembly and energy transfer (3). Evaluation of the molecular mechanisms underlying degradation of the phycobilisome under stress conditions employs mutants that, unlike wild-type cells, do not degrade their pigments during nitrogen and sulfur deprivation. Such mutants have been thus far isolated by a visual screen (4–7). Several drawbacks are inherent to this method, and it is also laborious and time-consuming. The visual screen is based on the observation that sulfur or nitrogen starvation triggers complete degradation of the phycobilisome (8). This results in a yellowish (bleached) color rather than the typical blue-green appearance of the cyanobacteria. To screen for mutants , mutagenized cells were plated on growth medium lacking added sulfur that was solidified with washed agar. Colonies growing on such medium, unless plated at a high density, were not starved enough to exhibit substantial pigment degradation. Therefore, this procedure resulted in very small and dense colonies that required screening under binoculars and several rounds of restreaking to obtain a clone exhibiting the desired phenotype. Furthermore, the rate of pigment degradation is affected by light intensity, and thus a color gradient appears in a single colony. This often leads to the isolation of false positives and generally makes it very difficult to obtain mutants exhibiting partial phenotypes; namely, those that degrade their pigments but to a lesser extent or at a slower rate compared to the wild-type strain. Here we describe the successful application of a fluorescence-activated cell sorter (FACS for the efficient isolation of mutants impaired in pigment degradation (nonbleaching mutants). The benefits of this technique can be amply demonstrated using the wild-type and the previously isolated nonbleaching mutant, nblRΩ. We analyzed the fluorescence properties of cells grown in complete and nutrient-depleted growth medium (Figure 1A). Fluorescence was measured following excitation at 590 nm, a wavelength that is efficiently absorbed by the cyano-bacterial light-harvesting complex. A broad fluorescence peak (610–700 nm) is observed in cells grown in complete medium. …