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Methods Appl Fluoresc. 2015 Mar 9;3(1):014005. doi: 10.1088/2050-6120/3/1/014005.

Correlative super-resolution imaging of RNA polymerase distribution and dynamics, bacterial membrane and chromosomal structure in Escherichia coli.

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1
Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt, Germany.

Abstract

We demonstrate correlative super-resolution PALM, PAINT and dSTORM imaging of RNA polymerase, membrane and chromosomal DNA in fixed E. coli. This protocol allows the combination of precise structural information of the nucleoid (dSTORM) with quantitative super-resolution imaging (PALM) of interacting proteins. The spatial distribution and organization of RNA polymerase and DNA are visualized in bacterial cells grown at doubling times of 25 or 44 min. We observe that RNA polymerase is concentrated at the edge of the highly structured nucleoid during fast growth, whereas it is found more evenly distributed during medium-fast growth. In both conditions, the nucleoid shows densely packed areas which appear to be inaccessible to RNA polymerase. This finding is confirmed by live-cell tracking of RNA polymerase and subsequent imaging of the respective nucleoids using a protocol for fast fixation on-the-slide.

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