Architecture of the Outer Membrane of Escherichia coli K12. Phase Transitions of the Bacteriophage K3 Receptor Complex

Abstract

The adsorption constant of the irreversible adsorption of the bacteriophage K3 to Escherichia coli K12 bacteria is strongly dependent on the incubation temperature. Two inflection points are observed in an Arrhenius plot. For cells grown at 37 "C the inflection points are found at 20 "C and 28 "C whereas these inflection points shift to 10 "C and 19 "C for cells grown at 12 "C.To study the lipid environment of the receptor the temperature dependence of the inactivation of bacteriophage K3 was measured in vitro in the presence of various lipids. The Arrhenius plots of the rate of inactivation of phage K3 by complexes of protein d and lipopolysaccharide are very similar to those observed for whole cells. With lipopolysaccharide isolated from cells grown at 37 "C inflection points are observed at 20 "C and 28 "C. With lipopolysaccharide from cells grown at 12 "C the inflection points are found at 10 "C and 21 "C. These results show that the environment of protein d in vivo can be mimicked perfectly in vitro by protein d/lipopolysaccharide complexes.The fatty acid composition of lipopolysaccharide isolated from cells grown at 37 "C and at 12 "C differs in that in the latter case the amounts of mono-unsaturated fatty acids (mainly palmitoleic acid) are increased at the expense of lauric acid. This difference in fatty acid composition probably explains the difference in the phase transition temperatures caused by the two lipopolysaccharide preparations. A transition at the inflection point of the highest temperature is also found for lipopolysaccharide using light-scattering measurements and appears to be a thermal transition, since it is also observed in differential scanning calorimetry.Cells of mutant strain CE1071 lacking outer membrane proteins b and c and concomitantly containing phospholipids in the outer leaflet of the outer membrane, adsorb phage K3 with an almost normal rate, but the shape of the Arrhenius plot differs from the curve of wild-type cells. The characteristics of the adsorption of phage K3 to these mutant cells can be mimicked in vitro by the incorporation of phospholipid into protein d/lipopolysaccharide complexes, indicating that phospholipids are part of the environment of the phage K3 receptor in cells of this mutant, but not in wildtype cellsThe outer membrane of gram-negative bacteria contains lipopolysaccharide, phospholipids and proteins. Lipopolysaccharide is located in the outer monolayer [l] whereas phospholipids are probably exclusively present in the inner monolayer [2,3]. One of the major proteins of the outer membrane is the heat-modifiable protein d [4,5], which is also referred to as protein 3a [6], 1I* [7] and 0-10 [8]. This protein has been purified in its non-heat modified form [7,9]. In complex with lipopolysaccharide this protein inactivates bacteriophages K3 [9] and TuII* [lo, 111. In the latter case the lipid A part of lipopolysaccharide is as active as intact lipopolysaccharide in generating phage receptor activity [l 11. Phospholipid or detergent cannot replace lipopol...

31P Nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. II. Lipopolysaccharide and lipopolysaccharide-phospholipid complexes

“…At 400(] (Fig. 6C), which is certainly above the phase transition as measured by light scattering [13], the position of the peaks was similar (the larger peak has shifted slightly to higher field}. The spectrum is much narrower, indicating more motion at 40°C possibly because the phase transition represents a gel-to-liquid crystalline transition [20].…”

“…4A). In this case the occurrence of stacked lamellae was not a result of phase segregation as glycerol is always present in these freeze-fracturing experiments but this morphology is probably a result of a temperature~lependent phase transition [13]. In addition to the stacked sheets, single sheets were observed (Fig.…”

“…Phospholipids were removed from the lipopolysaccharide preparations by repeated extractions with chloroform/methanol (2 : 1). No RNA could be detected in the sample [13]. This lipopolysaccharide is called native lipopolysaccharide.…”

“…Native lip0polysaccharide of E. coli K12 shows a phase transition from 28--31°C as measured by light scattering [13]. Therefore the freeze-fracture morphology of native and electrodialyzed (sodium salt) lipopolysaccharide was studied below and above this phase transition temperature by quenching the samples from 0 or 22°C or from 37°C, respectively.…”

“…2). Below the phase transition temperature (at 0 and 22°C) [13] thin rod-or ribbon-like structures with a length of approx. 200 nm were observed.…”