Several ␤-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the ␤-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the ␤-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution.␤-barrel-type channels are involved in the translocation of polypeptides (1), the assembly of proteins in the outer membrane of endosymbiotic organelles (2-4), or in the assembly of proteins in the outer membrane of bacteria (5, 6). These proteins belong to one class, which can be termed polypeptide-transporting ␤-barrel channels (2, 4, 7). Four proteins are in the focus of recent investigation, namely the bacterial outer membrane proteins Omp85 and ShlB, the mitochondrial outer membrane protein Tob55/Sam50, and the chloroplast outer envelope protein Toc75.ShlB is an outer membrane protein involved in the secretion of hemolysins or adhesins in various Gram-negative pathogens (8, 9). Omp85 is an essential component for outer membrane biogenesis in Neisseria meningitidis that might have two functions: the assembly of outer membrane proteins (5) and the translocation of lipids (10). Recently, it was discussed that the effect on lipid transfer by Omp85 depletion might be indirect and explained by an assembly defect of the required outer membrane protein, suggesting a function of Omp85 in outer membrane protein assembly only (11). As for ShlB, a ␤-barrel transmembrane structure was suggested for Omp85 (5). Recently, a new polypeptide-transporting protein was identified in the outer membrane of mitochondria and termed Sam50 (3), Tob55 (2), or mitochondrial Omp85 homologue (4). This protein facilitates the assembly of proteins into the outer membrane of mitochondria. Tob55/Sam50 is found in a larger complex with Mas37 (3, 12) and Tob38/Sam35 (13-15).The fourth investigated ␤-barrel-type polypeptide transporter is the 75-kDa subunit of the translocon of the outer envelope of chloroplasts, Toc75. Toc75 forms a complex with Toc34, Toc64, and Toc159 (16). In contrast to the other identified polypeptide transporters, such as Omp85, the translocation of proteins through Toc75 requires the action of assisting proteins, such as Toc159 (17), but still Toc75 seems to contain a preprotein-binding site as determined by electrophysiological measurements (1). Topological modeling of Toc75 from Pisum sativum (18) or T...

Anabaena is a model to analyze the evolutionary development of plastids, cell differentiation, and the regulation of nitrogen fixation. Thereby, the outer membrane proteome is the place of sensing environmental differences and during plastid development, systems for intracellular communication had to be added to the proteome of this membrane. We present a protocol for the isolation of the outer membrane from Anabaena and the analysis of the proteome using different tools. 55 proteins were identified.

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

Transport of solutes or macromolecules such as proteins across membranes requires a proteinaceous channel or transporter. Besides their way of action, these proteins can be divided according to their substrates or to their secondary structure of the membrane domain. In terms of secondary structure a-helical or b-sheet channels can be differentiated [1]. Both types of channels show a high neighbourhood correlation according to the fold [2] suggesting similar folds of the membrane inserted domains. In the past, much attention was given to the a-helical channels [3-5]. However, recently ion channels formed by the b-sheets moved into the focus of interest [6,7]. While analyzing these channels it became obvious that they emerged from outer membrane proteins of prokaryotic endo-symbionts, as these proteins were the only b-barrel type membrane proteins found in bacteria [6]. This class of proteins is present in organellar membranes of eukaryotic organisms, like in the outer mitochondrial membrane [8] emerged from a-proteobacteria [9], in the outer envelope of chloroplasts [10,11] emerged from cyanobacteria [9] and maybe even in the peroxi-somal membrane [12]. The peroxisomal b-barrel protein might be an indication either of the discussed endosymbiotic origin (for example [13]) or of a redistribution of proteins within the cell as a result of the gene transfer of the other two endosymbiotic events [14]. Most of the b-barrel type channels of eukaryotes belong to the porin type family. Recent research revealed that b-barrel type channels are also involved in the translocation of polypeptides [15], in the assembly of proteins in the outer membrane of endosymbio-tic organelles [16-19] or in the assembly of proteins in the outer membrane of bacteria [7,20,21]. One polypeptide-transporter that was found in Bordetella pertussis is FhaC, which secretes the main Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the b-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organ-elles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the b-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed. Abbreviation EBS, exact b-sheet.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

The multicellular Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N(2) in differentiated cells called heterocysts, which exchange nutritional and regulatory compounds with the neighbour photosynthetic vegetative cells. The outer membrane of this bacterium is continuous along the filament defining a continuous periplasmic space. The Anabaena alr0075, alr2269 and alr4893 gene products were characterized as Omp85-like proteins, which are generally involved in outer membrane protein biogenesis. Open reading frame alr2269 is the first gene of an operon that also carries genes for lipopolysaccharide lipid A biosynthesis including alr2270 (an lpxC homologue). Strains carrying inactivating alr2269 or alr2270 constructs showed enhanced sensitivity to erythromycin, SDS, lysozyme and proteinase K suggesting that they produce an outer membrane with increased permeability. These strains further exhibited increased uptake of sucrose, glutamate and, to a lesser extent, a few other amino acids. Increased uptake of the same metabolites was obtained by mechanical fragmentation of wild-type Anabaena filaments. These results document that the outer membrane is a permeability barrier for metabolites such as sucrose and glutamate, which are subjected to intercellular exchange in the diazotrophic filament of heterocyst-forming cyanobacteria.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N 2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane ␤-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).Gram-negative bacteria use a type I export system to transfer proteins or other molecules, like siderophores or fatty acids, to the cell surface (37, 50, 55). The proteinaceous substrates contain a C-terminal secretion signal essential and sufficient for export. The information for targeting and translocation, however, seems to be presented in the form of a secondary structure element rather than in the primary sequence as no clear amino acid motif has been identified. The machinery of type I export systems bridges the periplasm, allowing transfer of their substrates beyond the outer membrane. The export system is composed of a translocase spanning the plasma membrane with an ␣-helical, pore-forming domain and a cytosolic domain energizing the translocation process. Additionally, an adaptor protein associates with the translocase and, after substrate association, induces an interaction with an outer membrane TolC-like exit tunnel to conduct the secretion. Whereas TolC-like proteins from gram-negative proteobacteria have been amply investigated (37,50,54,55), nothing is known about these systems in cyanobacteria. Based on sequence similarity (40) and proteomic analysis of the cell wall fraction (42), we have recently proposed the existence of a protein of this type, Alr2887, in Anabaena sp. strain PCC 7120, for which a function in lipid transfer has previously been proposed (40).Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium which starts a program of cell differentiation upon nitrogen starvation that results in the appearance of nitrogen-fixing cells. These cells are called heterocysts and are arranged in a semiregular pattern along the filament (63). Three mechanisms protect the oxygen-labile nitrogenase from irreversible inactivation by O 2 produced by oxygenic photosynthesis: (i) formation of extra envelope layers outside the gram-negative cell wall, (ii) degradation of photosystem II antennae and a d...

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.