Estudi dels carotenoides en espècies marrons de bacteris verds del sofre: diversitat, eco-fisiologia i regulació

This study is focused on the composition, distribution and function of carotenoids in the photosynthetic antenna of brown-coloured species of Green Sulfur Bacteria (GSB). The first part of the work has focused on the development of two different reverse-phase high performance liquid chromatography (HPLC) methods to study the diversity of carotenoids from different species of GSB (Chapter 3). We also introduced a pre-treatment of samples that provides a better resolution on the separation of all carotenoids including the all-trans and cis isomers, in a run time of 45 min. For complex pigment samples, such as untreated extracts or sediment pigment samples where a high diversity of pigments is suspected, we modified an existing method (Borrego and Garcia-Gil, 1994) to achieve the complete separation of all carotenoids as well as all the different bacteriochlorophylls (BChls) homologues and algal pigments. This modification was successfully applied for the detection and identification of pigments in ancient sediments (0.71.5 million years-old) from a quarry located in the lacustrine area of Banyoles. The detection of signature pigments for brown-coloured GSB confirmed the presence of GSB in the lacustrine area of Banyoles at least since mid-Pleistocene. In this first part we also analyzed the carotenoids from Chlorobium (Chl) phaeobacteroides CL1401 by liquid chromatography coupled to mass spectrometry (LC-MS/MS) to confirm their identification and determine their molecular mass. Besides, several experiments were done to evaluate the effect of temperature, light and different oxidising and reducing chemical agents in the quantitative and qualitative composition of carotenoids and BCls in Chl. phaeobacteroides CL1401. These results confirmed that BCls are easily photodamaged and that the all-trans and cis isomers are constitutive components of the photosynthetic apparatus of GSB and then they are not artefacts produced during sample manipulation. Chapter 4 compiles a series of experiments carried out to elucidate the dynamics of pigment synthesis during growth of several species of GSB under different light conditions. Also these investigations allowed us to monitor the changes in the number of reaction centers (RC) during the light adaptation process. The number of RC was determined from the chromatographic quantification of BChl663, the primary acceptor in the electron transport chain in GSB. The estimation of RC per chlorosome was done according to both previous pigment stoichiometries and biometric values and from the experimental data obtained in this work. An average value of 70 RC per chlorosome was obtained. This value agrees with current models of pigment composition in the photosynthetic apparatus of GSB. In this chapter the study of the evolution of trans/cis ratios for the main carotenoids in GSB during light adaptation is also included. This ratio was calculated for different strains and chlorosomes of GSB and also from natural populations of Chl. phaeobacteroides thriving in a meromictic lake. No differences were found for the trans/cis ratio between cultures grown at saturating and limiting light conditions (trans/cis ratio of 2). Furthermore, similar values were found in isolated chlorosomes of different brown-coloured strains of GSB and in natural populations. In this case the trans/cis ratio remained fairly constant throughout the water column and also along the studied growth period of the population. Finally, in Chapter 5 we present a new molecular marker for the specific detection of brown-species of GSB. Although the designed primers originally targeted a gene involved in the biosynthetic pathway of carotenoids (CrtY that encodes a lycopene cyclase), final sequence retrieved matches with a gene related to the Polyketide ketosynthases protein family. The precise function of this protein in GSB remains hitherto unknown. However, the availability of this molecular tool opens the door for future investigations on the microbial ecology of brown-coloured species of GSB using techniques as Real-time PCR to selectively monitor populations of these microorganisms in their natural habitats.