Low-temperature-induced changes in composition and fluidity of lipopolysaccharides in the antarctic psychrotrophic bacterium Pseudomonas syringae

Abstract

The Antarctic psychrotrophic bacterium Pseudomonas syringae was more sensitive to polymyxin B at a lower (4 degrees C) temperature of growth than at a higher (22 degrees C) temperature. The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature. These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.

FIG. 1.

OM permeability of P. syringae cells as measured by NPN fluorescence assay. The fluorescence measurements were carried out at room temperature in the suspension of 4°C- and 22°C-grown cells (OD₆₀₀ = 0.5) containing 10 μM NPN and in the presence of a variable amount of polymyxin B. The degree of changes in the OM has been shown as a function of fluorescence intensity at 410 nm in arbitrary units (a.u.).

FIG. 2.

LPS profile of P. syringae grown at 4° (lane 1) and 22°C (lane 2). The LPS were isolated by the method of Brade and Galanos (1), separated on by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, and stained with silver nitrate (30).

FIG. 3.

Fluidity in the LPS aggregates as measured by the fluorescence emission spectra of pyrene. The purified LPS from 4° and 22°C-grown cells were extensively dialyzed against Milli-Q water and were pelleted by ultracentrifugation (100,000 × g, 4 h) to remove divalent cations. The lyophilized pellets of LPS were suspended in a buffer (5 mM HEPES, pH 7.4, and 150 mM NaCl), and the fluorescent probe pyrene (6.5 μM) was added to the suspension for recording fluorescence spectra (excitation at 333 nm and emission at 360 to 520 nm). The amount of monomer and dimer (excimer) of the pyrene molecules in the LPS aggregates was calculated from the fluorescence intensities at 372 and 470 nm, respectively, and the ratios were plotted. All measurements were carried out at room temperature.