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.

β-barrel-shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell-wall of Gram-negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane-localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to ana Omp85. Ana Tic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N-terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline-rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed. Abbreviations: BAM, β-barrel assembly machinery; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; OM, outer membrane; OMP, OM proteins; PC, phosphatidylcholine; PG, phosphatidylglycerol; PGL, peptidoglycan layer; PM, plasma membrane; POTRA, polypeptide-transportassociated domains; SQDG, sulfoquinovosyldiacylglycerol; TIC, translocon of the inner envelope membrane of chloroplasts.

The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. Those belong to the family of the two-partner secretion system proteins which are characteristic for pathogenic bacteria. Because pathogenicity of Anabaena sp. PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659, while TpsB2 is encoded by all5116. The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene-cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. PCC 7120 putatively coding for substrates of the TpsB-system suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes, respectively, resulted in phenotypes of altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. PCC 7120 and predict a large pool of putative substrates. IMPORTANCE Cyanobacteria are important organisms for the ecosystem considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacteria overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment including other organisms is required to define their impact on ecosystems. While two-partner secretion systems are well known from pathogenic bacteria, we provide a first description of the cyanobacterial two-partner secretion system.

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