Ultrastructural Study of Salmonella typhimurium Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components

Abstract

Amino groups of cell envelope proteins, lipids and lipopolysaccharides cannot be labeled in intact cells of S. typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. Application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-HCl, EDTA (Na salt) (EDTA), divalent cations and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a MW of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B+ and of proteins with MW of 26,000, 22,000 and below 17,000. A combination of Tris-HCl and EDTA induced additional dansylation of the major protein A and of proteins of MW 80,000, 60,000 and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B+ was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. The peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-HCl-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.