Incorporation of cyclodiene pesticides and their polar metabolites to model membranes of soil bacteria

The world-wide application of cyclodiene pesticides (CP) lead to severe pollution of arable land and because of the long half-lives they will be for many decades present in the soil. The only reasonable way of the elimination of these chemicals from the soil is bioremediation – the introduction to the soil of decomposer microorganisms strains capable of CP degradation. CP are highly hydrophobic and exhibit large membrane activity; thus, they can be incorporated to the cellular membrane and retained therein. The presence of CP and their metabolites in the cellular membrane of the decomposer organism can lead to severe alterations of its function and in consequence to the death of the decomposer cell. Microorganisms protect themselves changing the phospholipid composition of their membranes. To shed light on the correlation between the membrane composition and its interactions with CP and their metabolites we applied Langmuir monolayers as versatile models of decomposers’ membranes. By the proper selection of phospholipids we prepared different models of cellular membranes of Gram-negative and Gram-positive bacteria. The model membranes were doped by four most frequently applied CP and their common metabolite. The combined application of microscopic, diffractometric and spectroscopic methods proved that CP can be incorporated into the model membranes and that the membrane activity of endosulfan is comparable with endrin – one of the most toxic pesticides. The penetration tests and spectroscopic studies proved also the possibility of the uptake of the polar CP metabolites by the model membranes from the aqueous subphase

The composition of phospholipid model bacterial membranes determines their endurance to secretory phospholipase A2 attack : the role of cardiolipin

Soil bacteria are decomposer organisms crucial for the biodegradation of organic pollutants, mineralization of dead organic matter and the turnover of biogenic elements. In their environment they are constantly exposed to membrane-lytic enzymes emitted to the soil by other microorganisms competing for the same niche. Therefore, the composition and structure of their membranes is of utmost importance for survival in the harsh environment. Although soil bacteria species can be Gram-negative or Gram-positive and their membranes differ significantly, they are formed by phospholipids belonging mainly to three classes: phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipins (CL). The correlation of the membrane phospholipid composition and its susceptibility to secretory membrane-lytic enzymes is widely unknown; thus, to shed light on these phenomena we applied the Langmuir monolayer technique to construct models of soil bacteria membranes differing in the mutual proportion of the main phospholipids. To characterize the systems we studied their elasticity, mesoscopic texture, 2D crystalline structure and discussed the thermodynamics of the interactions between their components. The model membranes were exposed to secretory phospholipase A2. It turned out that in spite of the structural similarities the model membranes differed significantly in their susceptibility to s-PLA2 attack. The membranes devoid of cardiolipin were completely degraded, whereas, these containing cardiolipin were much more resistant to the enzymatic hydrolysis. It also turned out that the sole presence of cardiolipin in the model membrane did not guarantee the membrane durability and that the interplay between cardiolipin and the zwitterionic phosphatidylethanolamine was here of crucial importance

Label-free infrared spectroscopy and imaging of single phospholipid bilayers with nanoscale resolution

Mid-infrared absorption spectroscopy has been used extensively to study the molecular properties of cell membranes and model systems. Most of these studies have been carried out on macroscopic samples or on samples a few micrometers in size, due to constraints on sensitivity and spatial resolution with conventional instruments that rely on far-field optics. Properties of membranes on the scale of nanometers, such as in-plane heterogeneity, have to date eluded investigation by this technique. In the present work, we demonstrate the capability to study single bilayers of phospholipids with near-field mid-infrared spectroscopy and imaging and achieve a spatial resolution of at least 40 nm, corresponding to a sample size of the order of a thousand molecules. The quality of the data and the observed spectral features are consistent with those reported from measurements of macroscopic samples and allow detailed analysis of molecular properties, including orientation and ordering of phospholipids. The work opens the way to the nanoscale characterization of the biological membranes for which phospholipid bilayers serve as a model