Fluorescence Studies on the Structure of Bacterial Envelopes

Abstract

The quantitative nutritional requirements for the growth of Pseudomonas cepacia NCTC 10661 were determined. The ability of P. cepacia to store iron was indicated. The electrophoretic mobilities of logarithmic and nutrient-depleted cells of P. cepacia decreased in the following order: logarithmic, carbon, oxygen, Sulphate, phosphate, nitrogen, iron, and magnesium-depleted cells. The surface hydrophobicity of the same cell types was determined using contact angles with cell layers, and the angles decreased as follows: phosphate, oxygen, nitrogen, carbon, magnesium, sulphate, logarithmic, and iron-depleted cells. The killing of logarithmic-phase cells (P. cepacia) by whole blood was increased when the temperature was increased. Increase in resistance to killing by whole blood was in the following order: carbon, iron, sulphate, nitrogen, phosphate, and magnesium-depleted cells. The permeability barrier of the outer membrane (OM) was disrupted by Tris-buffer as studied by fluorescent probes. The change of the envelope of P. cepacia at different stages of the growth curve was studied with 3,3’-dipentyloxacarbocyanine iodide. Fluorescence intensity was highest at the turn-off point and then decreased depending on the nutrient being depleted. The OM was three times as viscous as the cytoplasmic membrane (CM) in magnesium and iron-depleted cells. Sarkosyl increased the microviscosity of the OM as studied with several fluorescent probes. Separation of the OM from the CM was performed by sucrose density gradient centrifugation. The OM protein profile of magnesium-depleted cells was much simpler than that of iron and oxygen-depleted cells. The synthesis of the protein with a molecular weight of 66,000 daltons was induced by iron depletion, and two OM proteins (17,000 and 18,500 daltons) were induced by oxygen depletion. The OM consisted of 50% protein and 20% phospholipid (PL), and the rest was probably LPS, while the CM consisted of 80% PL and 20% protein. The OM was enriched in phosphatidylethanolamine, while the CM was enriched in phosphatidylglycerol.