Structure and Function of a Bacterial Gap Junction Analog.

Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.

Heterocyst Septa Contain Large Nanopores That Are Influenced by the Fra Proteins in the Filamentous Cyanobacterium Anabaena sp. Strain PCC 7120.

Multicellular heterocyst-forming cyanobacteria, such as Anabaena, grow as chains of cells forming filaments that, under diazotrophic conditions, contain two cell types: vegetative cells that perform oxygenic photosynthesis and N2-fixing heterocysts. Along the filament, the intercellular septa contain a thick peptidoglycan layer that forms septal disks. Proteinaceous septal junctions connect the cells in the filament traversing the septal disks through nanopores. The fraCDE operon encodes proteins needed to make long filaments in Anabaena. FraC and FraD, located at the intercellular septa, are involved in the formation of septal junctions. Using a superfolder-green fluorescent protein (GFP) fusion, we found in this study that FraE is mainly localized to the poles of the heterocysts, consistent with the requirement of FraE for constriction of the heterocyst poles to form the "heterocyst neck." A fraE insertional mutant was impaired by 22% to 38% in transfer of fluorescent calcein from vegetative cells to heterocysts. Septal disks were inspected in murein sacculi from heterocyst-enriched preparations. Unexpectedly, the diameter of the nanopores in heterocyst septa was about 1.5- to 2-fold larger than in vegetative cell septa. The number of these nanopores was 76% and 6% of the wild-type number in fraE and fraC fraD mutants, respectively. Our results show that FraE is mainly involved in heterocyst maturation, whereas FraC and FraD are needed for the formation of the large nanopores of heterocyst septa, as they are for vegetative cell nanopores. Additionally, arrays of small pores conceivably involved in polysaccharide export were observed close to the septal disks in the heterocyst murein sacculus preparations. IMPORTANCE Intercellular communication, an essential attribute of multicellularity, is required for diazotrophic growth in heterocyst-forming cyanobacteria such as Anabaena, in which the cells are connected by proteinaceous septal junctions that are structural analogs of metazoan connexons. The septal junctions allow molecular intercellular diffusion traversing the septal peptidoglycan through nanopores. In Anabaena the fraCDE operon encodes septal proteins involved in intercellular communication. FraC and FraD are components of the septal junctions along the filament, whereas here we show that FraE is mainly present at the heterocyst poles. We found that the intercellular septa in murein sacculi from heterocysts contain nanopores that are larger than those in vegetative cells, establishing a previously unknown difference between heterocyst and vegetative cell septa in Anabaena.

Pentapeptide-repeat, cytoplasmic-membrane protein HglK influences the septal junctions in the heterocystous cyanobacterium Anabaena.

N2 -fixing heterocystous cyanobacteria grow as chains of cells that are connected by proteinaceous septal junctions, which traverse the septal peptidoglycan through nanopores and mediate intercellular molecular transfer. In the model organism Anabaena sp. strain PCC 7120, proteins SepJ, FraC and FraD, which are localized at the cell poles in the intercellular septa, are needed to produce septal junctions. The pentapeptide-repeat, membrane-spanning protein HglK has been described to be involved in the deposition of the heterocyst-specific glycolipid layer, but the hglK mutant also showed intercellular septa broader than in the wild type. Here we found that hglK mutant of Anabaena is impaired in the expression of heterocyst-related genes coxB2A2C2 (cytochrome c oxidase) and nifHDK (nitrogenase), indicating a defect in heterocyst differentiation. HglK was predominantly localized at the intercellular septa and was required to make long filaments, produce a normal number of nanopores and express full intercellular molecular transfer activity. However, the effects of hglK inactivation were not additive to those of the inactivation of sepJ and/or fraC-fraD. We suggest that HglK contributes to the architecture of the intercellular septa with an impact on the function of septal junctions.

A nanopore array in the septal peptidoglycan hosts gated septal junctions for cell-cell communication in multicellular cyanobacteria.

Some filamentous cyanobacteria are phototrophic bacteria with a true multicellular life style. They show patterned cell differentiation with the distribution of metabolic tasks between different cell types. This life style requires a system of cell-cell communication and metabolite exchange along the filament. During our study of the cell wall of species Nostoc punctiforme and Anabaena sp. PCC 7120 we discovered regular perforations in the septum between neighboring cells, which we called nanopore array. AmiC-like amidases are drilling the nanopores with a diameter of 20 nm, and are essential for communication and cell differentiation. NlpD-like regulators of AmiC activity and septum localized proteins SepJ, FraC and FraD are also involved in correct nanopore formation. By focused ion beam (FIB) milling and electron cryotomography we could visualize the septal junctions, which connect adjacent cells and pass thru the nanopores. They consist of cytoplasmic caps, which are missing in the fraD mutant, a plug inside the cytoplasmic membrane and a tube like conduit. A destroyed membrane potential and other stress factors lead to a conformational change in the cap structure and loss of cell-cell communication. These gated septal junctions of cyanobacteria are ancient structures that represent an example of convergent evolution, predating metazoan gap junctions.

The role of FraI in cell-cell communication and differentiation in the hormogonia-forming cyanobacterium Nostoc punctiforme.

Multicellular cyanobacteria, like Nostoc punctiforme, rely on septal junctions for cell-cell communication, which is crucial for coordinating various physiological processes including differentiation of N2-fixing heterocysts, spore-like akinetes, and hormogonia-short, motile filaments important for dispersal. In this study, we functionally characterize a protein, encoded by gene Npun_F4142, which in a random mutagenesis approach, initially showed a motility-related function. The reconstructed Npun_F4142 knockout mutant exhibits further distinct phenotypic traits, including altered hormogonia formation with significant reduced motility, inability to differentiate heterocysts and filament fragmentation. For that reason, we named the protein FraI (fragmentation phenotype). The mutant displays severely impaired cell-cell communication, due to almost complete absence of the nanopore array in the septal cell wall, which is an essential part of the septal junctions. Despite lack of communication, hormogonia in the ΔfraI mutant maintain motility and phototactic behavior, even though less pronounced than the wild type (WT). This suggests an alternative mechanism for coordinated movement beyond septal junctions. Our study underscores the significance of FraI in nanopore formation and cell differentiation, and provides additional evidence for the importance of septal junction formation and communication in various differentiation traits of cyanobacteria. The findings contribute to a deeper understanding of the regulatory networks governing multicellular cyanobacterial behavior, with implications for broader insights into microbial multicellularity. IMPORTANCE: The filament-forming cyanobacterium Nostoc punctiforme serves as a valuable model for studying cell differentiation, including the formation of nitrogen-fixing heterocysts and hormogonia. Hormogonia filaments play a crucial role in dispersal and plant colonization, providing a nitrogen source through atmospheric nitrogen fixation, thus holding promise for fertilizer-free agriculture. The coordination among the hormogonia cells enabling uniform movement toward the positive signal remains poorly understood. This study investigates the role of septal junction-mediated communication in hormogonia differentiation and motility, by studying a ΔfraI mutant with significantly impaired communication. Surprisingly, impaired communication does not abolish synchronized filament movement, suggesting an alternative coordination mechanism. These findings deepen our understanding of cyanobacterial biology and have broader implications for multicellular behavior in prokaryotes.

The role of SepF in cell division and diazotrophic growth in the multicellular cyanobacterium Anabaena sp. strain PCC 7120.

The cyanobacterium Anabaena forms filaments of cells that grow by intercalary cell division producing adjoined daughter cells connected by septal junction protein complexes that provide filament cohesion and intercellular communication, representing a genuine case of bacterial multicellularity. In spite of their diderm character, cyanobacterial genomes encode homologs of SepF, a protein normally found in Gram-positive bacteria. In Anabaena, SepF is an essential protein that localized to the cell division ring and the intercellular septa. Overexpression of sepF had detrimental effects on growth, provoking conspicuous alterations in cell morphology that resemble the phenotype of mutants impaired in cell division, and altered the localization of the division-ring. SepF interacted with FtsZ and with the essential FtsZ tether ZipN. Whereas SepF from unicellular bacteria generally induces the bundling of FtsZ filaments, Anabaena SepF inhibited FtsZ bundling, reducing the thickness of the toroidal aggregates formed by FtsZ alone and eventually preventing FtsZ polymerization. Thus, in Anabaena SepF appears to have an essential role in cell division by limiting the polymerization of FtsZ to allow the correct formation and localization of the Z-ring. Expression of sepF is downregulated during heterocyst differentiation, likely contributing to the inhibition of Z-ring formation in heterocysts. Finally, the localization of SepF in intercellular septa and its interaction with the septal-junction related proteins SepJ and SepI suggest a role of SepF in the formation or stability of the septal complexes that mediate cell-cell adhesion and communication, processes that are key for the multicellular behavior of Anabaena.

Coexistence of Communicating and Noncommunicating Cells in the Filamentous Cyanobacterium Anabaena.

In filamentous heterocyst-forming (N2-fixing) cyanobacteria, septal junctions join adjacent cells, mediating intercellular communication, and are thought to traverse the septal peptidoglycan through nanopores. Fluorescence recovery after photobleaching (FRAP) analysis with the fluorescent marker calcein showed that cultures of Anabaena sp. strain PCC 7120 grown in the presence of combined nitrogen contained a substantial fraction of noncommunicating cells (58% and 80% of the tested vegetative cells in nitrate- and ammonium-grown cultures, respectively), whereas cultures induced for nitrogen fixation contained far fewer noncommunicating cells (16%). A single filament could have communicating and noncommunicating cells. These observations indicate that all (or most of) the septal junctions in a cell can be coordinately regulated and are coherent with the need for intercellular communication, especially under diazotrophic conditions. Consistently, intercellular exchange was observed to increase in response to N deprivation and to decrease rapidly in response to the presence of ammonium in the medium or to nitrate assimilation. Proteins involved in the formation of septal junctions have been identified in Anabaena and include SepJ, FraC, and FraD. Here, we reevaluated rates of intercellular transfer of calcein and the number of nanopores in mutants lacking these proteins and found a strong positive correlation between the two parameters only in cultures induced for nitrogen fixation. Thus, whereas the presence of a substantial number of noncommunicating cells appears to impair the correlation, data obtained in diazotrophic cultures support the idea that the nanopores are the structures that hold the septal junctions.IMPORTANCE Multicellularity is found in bacteria as well as in eukaryotes, and the filamentous heterocyst-forming (N2-fixing) cyanobacteria represent a simple and ancient paradigm of multicellular organisms. Multicellularity generally involves cell-cell adhesion and communication. The cells in the cyanobacterial filaments are joined by proteinaceous septal junctions that mediate molecular diffusion. The septal junctions traverse the septal peptidoglycan, which bears holes termed nanopores. Our results show that the septal junctions can be coordinately regulated in a cell and emphasize the relationship between septal junctions and nanopores to build intercellular communication structures, which are essential for the multicellular behavior of heterocyst-forming cyanobacteria.

Septal protein SepJ from the heterocyst-forming cyanobacterium Anabaena forms multimers and interacts with peptidoglycan.

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long. Proteinaceous septal junctions provide cell-cell binding and communication functions in the filament. In Anabaena sp. strain PCC 7120, the SepJ protein is important for the formation of septal junctions. SepJ consists of integral membrane and extramembrane sections - the latter including linker and coiled-coil domains. SepJ (predicted MW, 81.3 kDa) solubilized from Anabaena membranes was found in complexes of about 296-334 kDa, suggesting that SepJ forms multimeric complexes. We constructed an Anabaena strain producing a double-tagged SepJ protein (SepJ-GFP-His10) and isolated the tagged protein by a two-step affinity chromatography procedure. Analysis of the purified protein preparation provided no indication of the presence of specific SepJ partners, but suggested that SepJ is processed to remove an N-terminal fragment. Additionally, pull-down experiments showed that His6-tagged versions of SepJ and of the SepJ coiled-coil domain interact with Anabaena peptidoglycan (PG). Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled-coil domain is involved in this interaction. These observations support the idea that SepJ is a component of the septal junctions that join the cells in the Anabaena filament.

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120.

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N2-fixing heterocysts and CO2-fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N-acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell-cell communication in Nostoc punctiforme. This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2, were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme, because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell-cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD.