Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Abstract

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying chargecoupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "livecell stains" (DRAQ5 and SYTO 61) and also 4=,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoidribosome segregation. Possible underlying causes are suggested.T he morphology of bacterial nucleoids (the regions containing the chromosomal DNA) is sensitive to the stage of growth (degree of chromosome replication and segregation), to the quality of growth medium, and to the application of external stresses such as nutrient downshift or treatment with drugs (1-5). Highresolution structural studies of bacterial nucleoids using electron microscopy (EM) on slices of fixed cells have a long history (6). Over time, it became clear that nucleoid morphology is highly sensitive to the method of fixation (6, 7). Wide-field fluorescence microscopy lacks the spatial resolution of EM, but it has the important advantage of enabling imaging of live cells as they grow. Live-cell imaging thus offers the possibility of monitoring timedependent changes in nucleoid morphology during normal growth or following application of an external stress.A simple, general method for nonspecific, nonperturbative fluorescent staining of DNA in live bacterial cells remains elusive. One useful strategy places a genetically encoded fluorescent protein on the broadly distributed DNA-binding protein HU (2, 8, 9). However, there is not yet a clear understanding of how HU and other DNA-binding proteins distribute across the chromosomal DNA. As a simple alternative strategy, in this study, we tested a variety of commercially available DNA-staining dyes for their utility in nucleoid imaging for live E. coli cells. When used in flow cytometry assays, these stains are often c...

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

“…2A) (29). The spatial distribution of mobile RNAPs in the cell closely followed that of DNA, with only a few excursions of RNAP tracks into the surrounding cytoplasm, indicating that most promotersearching RNAP molecules remain within the volume of the nucleoid (Fig.…”

Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements

“…A detailed understanding of how fluorescent dyes bind with host DNA may prove essential to drug development efforts [48], or DNA curtains studies [49]. Using single-molecule orientation measurements, we characterized the dye SYTOX Orange—a DNA stain that has recently shown promise for live cell imaging applications [50]. While the chemical structure of SYTOX Orange is proprietary, this dye is reported to be monomeric, and to intercalate when binding to DNA (dye molecules are expected to slot between adjacent base pairs, orienting roughly perpendicular to the DNA axis) [51].…”

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

“…In a different vein, well established single-particle tracking methods would enable studies of AMP effects on the spatial distribution and movement of key cytoplasmic actors. Possibilities include DNA morphology and local diffusive motion [26, 27], and the spatial distribution and biochemical activity of ribosomes and RNA polymerase [23, 39, 40], among many others.…”