Response of Microcystis aeruginosa PCC 7806 to different CO2 concentrations

Future climatic change scenarios predict rising of the atmospheric CO2 levels which could favor the proliferation of some harmful bloom-forming cyanobacteria as Microcystis aeruginosa. In the present study, the response of M. aeruginosa strain PCC 7806 to two different partial pressure of CO2 was tested. Sandrini et al. (2013) recently found that several, but not all, M. aeruginosa strains lack the SbtA or BicA HCO3- uptake system genes; the contribution of different Ci transporters to photosynthesis and the difference between low and high affinity activated Ci uptake state were investigated. M. aeruginosa PCC 7806 was cultured in four chemostats containing modified BG11 medium with 10 mM NaNO3 and no presence of NaCl, NaHCO3, Na2CO3 and additional buffers. A wide variety of analysis on samples collected from continuous cultures – such as A750, medium composition, cellular composition, cell counting, mini-PAM, measurements with the O2 optode, Aminco, 77K fluorescence emission spectra – was carried out. Data analysis results showed that the increased CO2 concentration has a big effect on M. aeruginosa PCC 7806. Experiments were performed using the Oxy-4 O2 optode apparatus in order to measure the photosynthetic O2 evolution of samples taken from both batch and chemostat cultures. At low bicarbonate concentration, an evident inhibition of Na+-dependent HCO3- transporter BicA by LiCl at 25 mM was observed. The consequent addition of 25 mM NaCl was able to counteract the Li+ effect at pH 8.0 but not at pH 10.0. In the latter case, only the addition of a higher amount of HCO3- led to photosynthetic O2 evolution suggesting the important role of the BicA transporter. However, further studies are needed to better explain the results obtained as high pH levels might have an influence on the transport systems, altering the mechanism of pH regulation and the functioning of Na+/H+ antiporter systems.

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.