Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus

Abstract

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle

“…act. 9.25 Gbq/mmol) [16] have already been described. 2-Phenylethanol was obtained from Sigma (USA).…”

“…As another advantage, ANno can be incorporated into the lipids of prokaryotic [14] and eukaryotic [15] cells at a high rate via regular lipid metabolic pathways, thus enabling constitutive lipids to be studied in situ. In the membrane of the gram-positive bacterium Micrococcus luteus, this approach coupled with a study of the transverse distribution of lipids revealed the existence of heterogeneities in the lateral distribution of the two major lipids, namely phosphatidylglycerol and dimannosyldiacylglycerol, [16]. These results were obtained with non-synchronously growing cells, harvested at the end of the exponential *Corresponding author.…”

“…luteus (ATCC 4698-4) was grown in PWYE peptone-containing culture medium at 30°C under agitation as previously described [16]. Absorbance of cell suspensions was measured with a spectrophotometer (model Lambda 16, Perkin-Elmer, UK) at a wavelength of 550 nm.…”

“…As previously described [16], 2-ml samples of the bacterial suspension (A550 = 5) were illuminated for 3 min at a temperature of ~ 30°C in a fluorescence cuvette disposed on an optical bench transmitting light (above 340 nm) from a 200-W mercury arc lamp. The illuminated cells were washed to remove residual anthracene fatty acid present in the cell wall and the lipids were extracted as described above.…”

“…The theoretical background for calculating the lipid distribution has been exposed in detail elsewhere [16]. Briefly, the lipid phase in a membrane can be considered as a collection of n molecules which distribute between xl,...xj,...xn molecules (xl + ...xj + ...xn = n) of M1,...Mj,...M, different molecular species, respectively.…”