Abstract

The filamentous cyanobacterium Anabaena sp. PCC 7120 (further referred to as Anabaena sp.) is a model system to study nitrogen fixation, cell differentiation, cell pattern formation and evolution of plastids. It is a multicellular photosynthetic microorganism consisting of two cell types, vegetative cells and nitrogen fixing heterocysts. This study focuses on the function and dynamics of the proteome of the Gram-negative outer membrane in Anabaena sp. with emphasis on cell differentiation and iron limitation. The newly developed methods for the membrane fractionation are presented, followed by analysis and comparison of the outer membrane proteomes of vegetative cells and heterocysts. The absence of major proteomic alterations in the outer membrane between two cell types, together with the presented data on GFP activity in mutant strains, experimentally support the previously proposed continuum of the outer membrane and the periplasm in Anabaena sp. filament. Also, somewhat different properties of the Anabaena sp. periplasm than in unicellular cyanobacteria are suggested. Furthermore, two common classes of the outer membrane -barrel proteins are analyzed closer. First, Alr2887 protein, as shown here, is a TolC homologue present in both cell types. Protein secretion through Alr2887 / TolC channel-tunnel is essential for the heterocysts maturation and the glycolipid layer formation. Furthermore, the inner membrane ABC transporter encoded by devBCA operon is proposed as component of the TolC efflux system in Anabaena sp. heterocysts. Second, phylogenetic analysis of the surprisingly abundant protein family of 24 TonB-dependent iron transporters in Anabaena sp. is presented. Five members of this family are detected in the outer membrane of vegetative cells under iron-repletion and two of them, All4026 and Alr0397, are explored closer. It is demonstrated that the function of these iron transporters is required for maintaining iron homeostasis of the filaments under iron-replete conditions. Consequently, their gene expression is constant and not enhanced by iron limitation. All4026 and Alr0397 have different specificity for siderophore substrates and in addition to iron transport, All4026 protein is capable of copper uptake and influence on copper homeostasis in Anabaena sp. as well.