Single bacterium proteomics: Analysis of the RecA protein in single Deinococcus radiodurans

Conventional bacterial proteomics techniques are generally limited by sensitivity, selectivity or throughput to the study of high-abundance proteins in bulk cultures. This dissertation presents a comprehensive set of tools to analyze protein expression in single bacteria. These tools are applied to the study of a DNA repair protein, RecA, in single Deinococcus radiodurans.;D. radiodurans is the world's most radiation-resistant organism. The RecA protein is essential for efficient and rapid DNA repair in these bacteria. Current literature reports the results of proteomic and transcriptomic studies on millions of bacteria; this dissertation addresses RecA expression in individual D. radiodurans.;Confocal microscopy and flow cytometry were used to generate a population distribution of RecA expression. These analyses show a subpopulation of D. radiodurans that expressed high levels of RecA. Sorting analysis of the subpopulations suggest stochastic expression of RecA, leading to a survivor phenotype.;To improve the sensitivity of flow cytometry analysis, a flow cytometer was developed on a capillary electrophoresis (CE) system. This instrument provides improvements in the detection limits of flow cytometry, with multicolor detection potential.;Capillary electrophoresis with laser-induced fluorescence (CE-LIF) was also applied for chemical cytometry of D. radiodurans. The chemical contents of single bacteria were separated in minutes using CE. LIF detection provided yoctomole detection of EGFP. Analysis of single D. radiodurans suggests that recA is expressed at low levels in most resting cells.;Advances in whole proteome fluorescent labeling for CE-LIF are also addressed. The current labeling strategy relies on the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). The concentration limit of derivatization for FQ prevents its use in single bacterium proteomics. The optical and chemical requirements for a replacement label are discussed, and a pseudo-fluorogenic dye, Py1, is presented as a potential replacement for FQ.;This dissertation presents several tools to characterize protein expression in single bacteria. No individual tool is currently capable of generating a comprehensive description of protein distribution across a population; instead, RecA expression in D. radiodurans was characterized using a combination of conventional and novel instrumentation.