Dissection fonctionnelle du métabolisme de l'amidon chez les cyanobactéries – starchevol

Scientific background and objectives : Starch consists of an elaborate semi-crystalline form of glucose storage found selectively in plants. Starch differs from bacterial or eukaryotic glycogen by its aggregation into large insoluble and partly crystalline granules. We have proposed that the crystalline state of these polymers was due to the selective splicing of misplaced chains within a hydrosoluble precursor. The phylogeny of the debranching enzyme form responsible for this splicing is clearly prokaryotic. However, up until a few months ago, no bacteria were ever reported to accumulate starch. Very recently, marine biologists have isolated from the North Atlantic ocean a strain accumulating carbohydrate granules whose shape and size were reminiscent of starch. We have axenized this strain (strain AY100521) and have made a preliminary characterization of its storage polysaccharide structure which was clearly related to starch. This strain is related to other diazotrophic unicellular cyanobacteria of group V that were very recently proven to accumulate semi-amylopectin, a polymer of intermediate structure between glycogen and amylopectin. All starch-accumulating cyanobacteria turned out to define diazotrophic single cell species. Nitrogenase being unable to fix nitrogen in the presence of oxygen, these unicellular organisms require a temporal separation of oxygenic photosynthesis from anoxic diazotrophy through circadian clock regulated mechanisms. Such a temporal separation of photosynthesis and diazotrophy has prompted the evolution of a new form of storage polysaccharide. The latter had to be able to pack tightly the large amounts of glucose required to fuel diazotrophy at night in an osmotically inert form. In addition, this novel polysaccharide should be less subjected to turnover during the photooxygenic light phase. We therefore hypothesize that starch is required for diazotrophy in single cell nitrogen-fixing cyanobacteria and that the plastid's ancestor was characterized by a physiology and genomic content very similar to those found in present day group V cyanobacteria. We propose to test these hypotheses and to investigate the nature of the starch synthesis pathway in group V cyanobacteria. We also wish to test the relatedness of these organisms to the plant plastids. Project description, methodology : We envision 4 different work-packages, 3 of which will deal with a detailed characterization, respectively, of the biochemical pathway of starch synthesis and breakdown, of the fine structure of cyanobacterial starch and of the relatiohship tying diazotrophy to carbohydrate storage through a functional mutant selection approach. A fourth work-package will deal with phylogenomic approaches aimed at testing the relatedness of the genes involved to those that are known to play functions in starch metabolism in the three distinct plant lineages (rhodophytes, glaucophytes and chlorophytes). We plan to use miniaturized techniques that we have been developing for the last two decades involving detailed investigations of enzyme content through zymogram techniques and partial purification of important enzymes which include, of course, the starch debranching enzyme. In order to study the genes involved in cyanobacterial starch synthesis, we will use the growing genomic resources (that include the full genome sequence of Crocosphaera watsonii, a cyanobacterium very closely related to strain AY100521). This sequence information will be used to feed our phylogenetic approach. On the other hand, we have successfully axenized and cloned strain AY100521 on solid media. Our preliminary mutant selection experiments clearly indicate that the iodine screening procedure for the selection of starch defective mutants is feasible. Expected results : The selection of mutants defective for starch biosynthesis will enable us to test whether the storage of glucose into large osmotically inert granules is required in unicellular cyanobacteria to feed diazotrophy. Mutant selection together with structural and enzymological characterization of the wild-type and mutant isogenic pairs will inform us on the nature and function of the starch metabolism pathway in cyanobacteria. Finally, our phylogenomic approach will provide information as to the relatedness of present day group V cyanobacteria to the plastid's ancestor.