A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels

Abstract

Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.

The effects of polydisperse crowders on the compaction of theEscherichia colinucleoid

“…We were interested in understanding if the above described nucleoid behaviors during rifampicin treatment also hold in moderately fast and slow growth conditions where ribosome and polysome concentrations are expected to be lower (Dai et al, ; Ehrenberg et al, ). Treating moderately fast growing cells with rifampicin, we observed an initial compaction of the nucleoid at about the 5 min timescale (Figure a–c), which is consistent with the earlier reports (Bakshi et al, ; Spahn et al, ). Although the effect of compaction in our experiments was smaller (3%–5%) (Figure a–c insets) than in the previous reports (about 8%), which were carried out in fast growth conditions, the presence of nucleoid contraction supports the idea of the existence of transertion linkages.…”

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

“…Furthermore, specific lipophilic, fluorogenic dyes can directly visualize the native membrane by reversible on-and off-binding in PAINT microscopy (ii) [14,26,28,29,42]. Also carbohydrates-targeting proteins like WGA and ConA or lipid-targeting probes like mCLING or FYVE can be used (iii) [15,37,41,53,54,57].…”

“…(d) Specific nucleotide sequences are labeled by single-molecule fluorescence in situ hybridization (smFISH) using short, complementary DNA/RNA oligomers (often designed as fluorogenic hairpins in a fluorophore-quencher combination) (i) [34]. DNA, in general, can be either visualized by the reversible on-and off-binding of fluorogenic DNA intercalator-dye constructs using DNA-PAINT (ii) [29] or by nuclear-associated proteins decorating it (iii) [46]. A method to label nascent DNA is by incorporation of the alkyne-modified thymidine analog EdU during DNA replication combined with subsequent click labeling (v) [26,27,29].…”

ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data

“…Finally, ColiCoords can optimize the coordinate system based on SMLM data of a membrane marker. This type of data can be obtained through various super-resolution techniques such as Point Accumulation for Imaging in Nanoscale Topography (PAINT) (27,(76)(77)(78), Stochastic Optical Reconstruction Microscopy (STORM) (79,80) or PhotoActivated Localization Microscopy (PALM) (81,82) imaging. The optimization process and a possible application thereof is illustrated in Figure 5.…”

“…Fluorescence microscopy has become a crucial tool in studying bacterial cell biology (1)(2)(3)(4)(5)(6)(7)(8). It is minimally invasive and allows for the study of living bacteria in a controlled environment and to monitor the motion and sub-cellular topologies of any proteinaceous factor (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) or nucleic acids (25)(26)(27)(28). Through the multitude of genetically programmable fluorescent protein probes (29)(30)(31)(32)(33)(34)(35)(36) and commercially available dyes (37)(38)(39)(40)(41) with different conjugation capabilities (42)(43)(44)(45)(46), virtually all components of the bacterial machinery can be studied with high specificity and spatio-temporal resolution.…”