Multiplicity and specificity of siderophore uptake in the cyanobacterium Anabaena sp. PCC 7120.

Many cyanobacteria secrete siderophores to sequester iron. Alternatively, mechanisms to utilize xenosiderophores have evolved. The overall uptake systems are comparable to that of other bacteria involving outer membrane transporters energized by TonB as well as plasma membrane-localized transporters. However, the function of the bioinformatically-inferred components is largely not established and recent studies showed a high diversity of the complexity of the uptake systems in different cyanobacteria. Thus, we approached the systems of the filamentous Anabaena sp. PCC 7120 as a model of a siderophore-secreting cyanobacterium. Anabaena sp. produces schizokinen and uptake of Fe-schizokinen involves the TonB-dependent transporter, schizokinen transporter (SchT), and the ABC-type transport system FhuBCD. We confirm that this system is also relevant for the uptake of structurally similar Fe-siderophore complexes like Fe-aerobactin. Moreover, we demonstrate a function of the TonB-dependent transporter IutA2 in Fe-schizokinen uptake in addition to SchT. The iutA2 mutant shows growth defects upon iron limitation, alterations in Fe-schizokinen uptake and in the transcription profile of the Fe-schizokinen uptake system. The physiological properties of the mutant confirm the importance of iron uptake for cellular function, e.g. for the Krebs cycle. Based on the relative relation of expression of schT and iutA2 as well as of the iron uptake rate to the degree of starvation, a model for the need of the co-existence of two different outer membrane transporters for the same substrate is discussed.

The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

UNLABELLED: Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1. IMPORTANCE: Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the mechanisms and components involved in bacteria are just emerging. Filamentous heterocyst-forming cyanobacteria behave as multicellular organisms and represent an excellent model to study prokaryotic cell-cell communication. A path for intercellular metabolite exchange appears to involve transfer through molecular structures termed septal junctions. They are reminiscent of metazoan gap junctions that directly link adjacent cells. In cyanobacteria, such structures need to traverse the peptidoglycan layers in the intercellular septa of the filament. Here we describe a factor involved in the formation of channels across the septal peptidoglycan layers, thus contributing to the multicellular behavior of these organisms.

Multiple modes of iron uptake by the filamentous, siderophore-producing cyanobacterium, Anabaena sp. PCC 7120.

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore- and non-siderophore-producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore-producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self-secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe') via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore- and non-siderophore-mediated iron uptake. While assimilation of Fe' and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe' reduction and uptake is advantageous for low-density cultures, while at higher densities siderophore uptake is preferred.

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Toc33 and Toc64-III cooperate in precursor protein import into the chloroplasts of Arabidopsis thaliana.

The import of cytosolically synthesized precursor proteins into chloroplasts by the translocon at the outer envelope membrane of chloroplasts (TOC) is crucial for organelle function. The recognition of precursor proteins at the chloroplast surface precedes translocation and involves the membrane-inserted receptor subunits Toc34 and Toc159. A third receptor, Toc64, was discussed to recognize cytosolic complexes guiding precursor proteins to the membrane surface, but this function remains debated. We analysed Arabidopsis thaliana plants carrying a T-DNA insertion in the gene encoding the Toc64 homolog Toc64-III. We observed a light intensity-dependent growth phenotype, which is distinct from the phenotype of ppi1, the previously described mutant of the TOC34 homolog TOC33. Furthermore, chloroplast import of the model precursor proteins pOE33 and pSSU into chloroplasts is reduced in protoplasts isolated from plants with impaired Toc64-III function. This suggests that Toc64-III modulates the translocation efficiency in vivo. A ppi1 and toc64-III double mutant shows a significant increase in the transcript levels of HSP90 and TOC75-III, the latter coding for the pore-forming TOC component. Remarkably, the protein level of Toc75-III is significantly reduced, suggesting that Toc64-III and Toc33 cooperate in the insertion or stabilization of Toc75-III. Accordingly, the results presented support Toc64 as an import-relevant component of the TOC complex.

Outer membrane continuity and septosome formation between vegetative cells in the filaments of Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a prototype filamentous nitrogen-fixing cyanobacterium, in which nitrogen fixation and photosynthesis are spatially separated. Recent molecular and cellular studies have established the importance of molecular exchange between cells in the filament, but the routes involved are still under investigation. Two current models propose either a continuous periplasm or direct connections between adjacent cells whose integrity requires the protein SepJ. We used electron tomography to analyze the ultrastructure of the septum between vegetative cells in the Anabaena filament and were able to visualize intercellular connections that we term 'SEPTOSOMES'. We observed that, whereas the existence of the septosome does not depend on the presence of SepJ, the spacing between the two plasma membranes of the septum was significantly decreased in a ΔsepJ mutant. In addition, we observed that the peptidoglycan layer of each cell enters the septum but the outer membrane does not. Thus, each cell in the filament is individually surrounded by a plasma membrane and a peptidoglycan layer, and physical cell-cell contacts are mediated by the septosome.