Iron uptake in Gram-negative bacteria involves four distinct steps: (i) siderophore synthesis, (ii) siderophore secretion into the extracellular space, (iii) iron chelation by the siderophores, and (iv) siderophore/iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. This process is well characterized for some proteobacterial systems, but largely unexplored and scarcely investigated in cyanobacteria such as the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Two putative siderophore synthesis clusters have been recently identified in this cyanobacterium. In addition, the export system for the main siderophore, schizokinen, secreted by Anabaena sp. PCC 7120 was described as well as the outer membrane transporter for its import from the extracellular space. We present the identification of components of three additional systems involved in siderophore-mediated iron uptake under iron-limiting conditions, namely TonB3, the ExbB3/ExbD3 and the Fhu systems. The transcription level of these genes is elevated under iron limitations and decreased under excess iron, while the expression levels of other members of these gene families and systems are impacted in distinct ways by other environmental conditions. Mutants of the tonB3, exbB3/exbD3 and fhu genes show an iron starvation phenotype. Thus, Anabaena sp. has a similar, yet distinct system for siderophore-dependent iron uptake compared with other proteobacteria.

SummaryIron 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.

Background:The metabolite and antibiotic export system of cyanobacteria is largely unexplored. Results: Uptake of ethidium bromid by Anabaena sp. depends on porin-type activity while its seceretion relies on HgdD. Conclusion:The antibiotic export of cyanobacteria involves a proton gradient-driven TolC activity and MFS-type proteins. Significance: TolC of Anabaena sp. is placed in the context of antibiotic uptake and export.

Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

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.

TonB dependent transporters (TBDT) are an essential protein family in bacteria involved in the uptake of a broad variety of molecules such as siderophore-chelated iron, which was the first described substrate. Meanwhile it is known that TBDTs are involved in the uptake of many metals, sugars and polypeptides. The action of TBDTs is regulated and energized by the plasma membrane anchored TonB, which is charged by a proton pump. The number of the genes coding for TBDTs varies in different species, which might reflect environmental adaptations or evolutionary variations of the system. For example, in the cyanobacterium Anabaena sp. PCC 7120 the large number of 22 genes coding for TBDTs has been identified and the expression of these genes has been explored in the absence of iron or copper as well as under nitrogen starvation. We describe the analysis of the expression of the TBDT genes and the according cytoplasmic-membrane localized components; the latter appear to have a lower degree of complexity in Anabaena sp. PCC 7120. This analysis unravels that the response is not sole dependent on the metal supply, but also on cell culture densities. In addition, we present a large group of FhuA-like genes which is expressed highest under standard conditions suggesting a function distinct from iron or copper transport. The genes are clustered according to the expression profile and the consequences for our understanding of the transport systems in Anabaena sp. PCC 7120 are discussed.

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.

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.