Structural and metabolic aspects of multicellularity in a heterocyst-forming cyanobacterium

Résumé

Multicellularity appears to have arisen several times during the course of evolution and has evolved in different phylogenetic groups, including cyanobacteria, a highly diverse group of oxygenic photosynthetic prokaryotes that exhibit a wide range of developmental processes. Cyanobacteria represent one of the most diverse prokaryotic phyla, with morphotypes ranging from unicellular to multicellular filamentous forms. Some filamentous cyanobacteria can produce different types of cells, each one with specific functions. In some cases, cell differentiation allows the filament to carry out tasks that are functionally incompatible, such as oxygenic photosynthesis and the fixation of atmospheric nitrogen. The general plan in this thesis was to address the study of some structural and metabolic aspects of multicellularity in the model heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Heterocyst-forming cyanobacteria grow as chains of cells (trichomes or filaments), and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. From a structural point of view, cyanobacteria are diderm bacteria, bearing two cellular membranes: the cytoplasmatic membrane and an outer membrane, the latter residing outside of the peptidoglycan layer (or murein sacculus). Filamentous cyanobacteria present a continuous outer membrane along the filament, determining the presence of a continuous periplasmic space that contains the peptidoglycan layer. Although the cell envelope from cyanobacteria has been studied in some detail, the role of this structure in multicellularity has not been addressed until recently. Chapter 1 of this thesis focuses on the possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, thus contributing to the growth of Anabaena forming long trichomes. In order to address this structural aspect of multicellularity in Anabaena sp. PCC 7120, a set of available peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes encoding class B penicillin-binding proteins isolated in this work have been used to study filament length and the response of their filaments to mechanical fragmentation. The results obtained indicate that alteration of both the peptidoglycan layer and the outer membrane influence filamentation, although none of these elements is as important as the septal protein SepJ. Because of the compartmentalization of photosynthetic CO2 fixation and N2 fixation processes in different cell types, an intercellular exchange of nutrients takes place in the cyanobacterial filament. Vegetative cells donate products of CO2 fixation, such as sucrose, glutamate and alanine to the heterocysts. Heterocysts, in turn, provide N2 fixation products to the vegetative cells, being glutamine a previously identified metabolite. However, the nitrogenous metabolites that are transferred from heterocysts to vegetative cells are still not fully known. The work presented in Chapter 2 aimed to study the role of cyanophycin, a biopolymer that serves as a dynamic nitrogen cellular reserve material, in the diazotrophic physiology, and of its derivative products as possible nitrogen vehicles in the diazotrophic filament of Anabaena. The results confirmed that ORF all3922 of Anabaena sp. PCC 7120 is the gene encoding isoaspartyl dipeptidase, the enzyme involved in the second step of cyanophycin degradation. Under diazotrophic conditions, the enzyme accumulates in vegetative cells, implying that the ß-aspartyl-arginine dipeptide produced by cyanophycinase in heterocysts is transferred intercellularly to the vegetative cells, where it would be hydrolysed releasing aspartate and arginine. Thus, the ß-aspartyl-arginine dipeptide has been identified as a nitrogen vehicle in the diazotrophic filament. Arginine appears to be an important metabolite in the physiology of cyanobacteria, not only because it is found in cyanophycin, but also because it might function as a nitrogen vehicle for intercellular molecular exchange, at least in part as ß-aspartyl-arginine. However, arginine catabolism is not well understood in these microorganisms and only few studies have been published regarding arginine catabolic enzymes. In Chapter 3 and Annex II of this thesis, the study of two genes, alr2310, encoding an ureohydrolase family protein, and alr4995, encoding a protein belonging to the guanidine-group modifying enzymes superfamily, has been addressed in order to investigate their possible roles in arginine catabolism. The results showed that Alr2310 is the speB gene of Anabaena sp. PCC 7120, encoding an agmatinase, which accumulates preferentially in vegetative cells during diazotrophic growth, and that its inactivation leads to a severe toxic effect that could result from interference of accumulated agmatine with heterocyst differentiation. On the other hand, Alr4995 is a novel enzyme that generates proline from arginine in a two-step reaction, with ornithine (or citrulline) as intermediate metabolite. The concluding remarks of this work is that the heterocyst-forming cyanobacteria present highly coordinated and unique features of compartmentalized metabolic pathways as a strategy of multicellular behavior.