title: 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 charge-coupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "live-cell 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 nucleoid-ribosome segregation. Possible underlying causes are suggested.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Somenath
authors.initials: S
authors.lastname: Bakshi
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Heejun
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Nambirajan
authors.initials: N
authors.lastname: Rangarajan
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Barns
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Bratton
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA Molecular Biophysics Program, University of Wisconsin-Madison, Madison, Wisconsin, USA weisshaar@chem.wisc.edu.
authors.forename: James C
authors.initials: JC
authors.lastname: Weisshaar
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects* / Q000254:growth & development; D002466:Cell Nucleolus / Q000737:chemistry / Q000187:drug effects*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects* / Q000254:growth & development; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling; D054804:Cathelicidins
chemical_list: D023181:Antimicrobial Cationic Peptides; D005456:Fluorescent Dyes; C031161:cecropin A; D054804:Cathelicidins