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ABSTRACT Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacteriumAnabaenasp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment ofAnabaena, the analysis of the extracellular metabolites and proteins of afurC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction ofAnabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome offurC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins andamiCgene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, includingamiC1andamiC2,could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in thefurC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments infurC-overexpressing cells, namely 1,6-anhydro-N-acetyl-β-D-muramic acid (anhydroMurNAc) and its associated disaccharide (β-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions.Anabaenasp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator inAnabaena, which modulates the response to several stresses. Here, we show thatfurC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer. 

The symbiosis between the diatom Hemiaulus hauckii and the heterocyst-forming cyanobacterium Richelia intracellularis makes an important contribution to new production in the world’s oceans, but its study is limited by short-term survival in the laboratory. In this symbiosis, R. intracellularis fixes atmospheric dinitrogen in the heterocyst and provides H. hauckii with fixed nitrogen. Here, we conducted an electron microscopy study of H. hauckii and found that the filaments of the R. intracellularis symbiont, typically composed of one terminal heterocyst and three or four vegetative cells, are located in the diatom’s cytoplasm not enclosed by a host membrane. A second prokaryotic cell was also detected in the cytoplasm of H. hauckii , but observations were infrequent. The heterocysts of R. intracellularis differ from those of free-living heterocyst-forming cyanobacteria in that the specific components of the heterocyst envelope seem to be located in the periplasmic space instead of outside the outer membrane. This specialized arrangement of the heterocyst envelope and a possible association of the cyanobacterium with oxygen-respiring mitochondria may be important for protection of the nitrogen-fixing enzyme, nitrogenase, from photosynthetically produced oxygen. The cell envelope of the vegetative cells of R. intracellularis contained numerous membrane vesicles that resemble the outer-inner membrane vesicles of Gram-negative bacteria. These vesicles can export cytoplasmic material from the bacterial cell and, therefore, may represent a vehicle for transfer of fixed nitrogen from R. intracellularis to the diatom’s cytoplasm. The specific morphological features of R. intracellularis described here, together with its known streamlined genome, likely represent specific adaptations of this cyanobacterium to an intracellular lifestyle.