One of the most useful augments to the basic technique is to measure cell lysis (Levina et al., 1999). This can be particularly helpful to distinguish between mechanisms of cell killing when one is comparing mutant channels with the native channel. Cells of E. coli K-12 lacking functional channels will lyse when subjected to a large hypoosmotic shock. Lysis is indicated by two parameters: decrease in the light scattering of the cell suspension (OD650) and increase in absorption (A26o/A28o) of the supernatant after centrifugation (full-speed Jouan microcentrifuge 30 s) to remove cells and cell debris (Levina et al., 1999). Others have modified this technique by adding ethidium bromide (0.5 mg/ml) to the shock medium (Powl et al., 2003). The effect is to intercalate the ethidium ions into the released DNA. Samples prepared in this way are stored in the dark for 45 min prior to centrifugation to remove cells and debris, and the fluorescence of the supernatant is measured by excitation at 254 nm and recording emission at 632 nm. One can systematically extend this method to any readily measurable assay for an enzyme that is released by cell lysis.
High-throughput fluorescence methods have also been described for identifying channel mutants (Maurer and Dougherty, 2001). In this development the cloned channel is expressed in E. coli MJF465 and the cells are subjected to various degrees of hypoosmotic shock into minimal medium containing propidium iodide (PI) and SYTO 9 dyes (Live/Dead BacLight™ assay). The samples are then incubated at 37° in an incubator. Both 96- and 384-well microtiter plate designs are used to read the samples in a fluorescence plate reader with excitation at 480 and 490 nm and emission at 500 and 635 nm, respectively, for PI and SYTO 9. The ratio of green/red (500/ 635 nm) plotted as a function of hypoosmotic shock correlates with the character of channel mutations (Maurer and Dougherty, 2001). The channel-free strain exhibits high green/red fluorescence until sufficient dilution is achieved to cause cell lysis. When the wild-type channel is introduced, a high green/red ratio is maintained until the highest dilution when activation of the MscL channels may lead to depolarization of the membrane, causing increased accumulation ofPI. Mutant channels exhibiting significant loss of function (LOF) show properties intermediate between the wild-type and the channel-free mutant. In contrast, cells expressing channels that possess gain-of-function (GOF) mutations exhibit low green/red ratios at all dilutions, suggesting that the inappropriate gating of the channels depolarizes the membrane, causing high PI uptake. This approach offers the potential for automated screening for mutant channels. How well the method deals with subtle phenotypes is not clear and there may be a need for a refinement of the assay to identify a range of mutants.
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