Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli.

摘要

Growing bacterial cells forming division septa have sites near the septa that are sensitive to EDTA shock. Cells treated with EDTA incorporate proteins and other molecules from the surrounding medium, probably via vesiclelike lesions at the septa that are induced by EDTA. The amount of protein taken up is proportional to the protein concentration in the permeabilization medium. Incorporated molecules equilibrate throughout the cytoplasm, and those with affinity for DNA bind to the nucleoid. Conditions that promote the viability of permeabilized cells and help to avoid otherwise irreversible effects of EDTA are defined. Procedures for selecting cells that have incorporated protein and for studying the distribution of the protein and its effects in growing-dividing cells are described. The procedure may have several applications to molecular and cellular biology; however, we describe here the localization in living cells of the histonelike protein HU. Fluorescence microscopy of cells containing different amounts of fluorescein-labeled HU (varied from approximately 10(3) to 10(5) molecules per cell) showed that the HU concentrates in the nucleoid and is uniformly distributed throughout this structure. Control experiments demonstrated that unlabeled interior parts of the nucleoid can be resolved when labeled proteins that do not bind DNA or enter the nucleoid are introduced into living cells. It was concluded that in vivo added HU binds primarily DNA and that there are no intrinsic restrictions on major regions of the nucleoid to which the added HU protein may bind.