Locations of membrane protein production in a cyanobacterium.

Cyanobacteria show an unusually complex prokaryotic cell structure including a distinct intracytoplasmic membrane system, the thylakoid membranes that are the site of the photosynthetic light reactions. The thylakoid and plasma membranes have sharply distinct proteomes, but the mechanisms that target proteins to a specific membrane remain poorly understood. Here, we investigate the locations of translation of thylakoid and plasma membrane proteins in the model unicellular cyanobacterium Synechococcus elongatus PCC 7942. We use fluorescent in situ hybridization to probe the locations of mRNAs encoding membrane-integral proteins, plus Green Fluorescent Protein tagging of the RplL subunit to reveal the location of ribosomes under different conditions. We show that membrane-integral thylakoid and plasma membrane proteins are translated in different locations. Thylakoid membrane proteins are translated in patches at the innermost thylakoid membrane surface facing the nucleoid. However, different proteins are translated in different patches, even when they are subunits of the same multiprotein complex. This implies that translation is distributed over the proximal thylakoid surface, with newly inserted proteins migrating within the membrane prior to incorporation into complexes. mRNAs encoding plasma membrane proteins form patches at the plasma membrane. Ribosomes can be observed at similar locations near the thylakoid and plasma membranes, with more ribosomes near the plasma membrane when conditions force rapid production of plasma membrane proteins. There must be routes for ribosomes and mRNAs past the thylakoids to the plasma membrane. We infer a system to chaperone plasma membrane mRNAs to prevent their translation prior to arrival at the correct membrane. IMPORTANCE Cyanobacteria have a complex and distinct membrane system within the cytoplasm, the thylakoid membranes that house the photosynthetic light reactions. The thylakoid and plasma membranes contain distinct sets of proteins, but the steps that target proteins to the two membranes remain unclear. Knowledge of the protein sorting rules will be crucial for the biotechnological re-engineering of cyanobacterial cells, and for understanding the evolutionary development of the thylakoids. Here, we probe the subcellular locations of the mRNAs that encode cyanobacterial membrane proteins and the ribosomes that translate them. We show that thylakoid and plasma membrane proteins are produced at different locations, providing the first direct evidence for a sorting mechanism that operates prior to protein translation.

CP12 fine-tunes the Calvin-Benson cycle and carbohydrate metabolism in cyanobacteria.

The regulatory protein CP12 can bind glyceraldehyde 3-phosphate dehydrogenase (GapDH) and phosphoribulokinase (PRK) in oxygenic phototrophs, thereby switching on and off the flux through the Calvin-Benson cycle (CBC) under light and dark conditions, respectively. However, it can be assumed that CP12 is also regulating CBC flux under further conditions associated with redox changes. To prove this hypothesis, the mutant Δcp12 of the model cyanobacterium Synechocystis sp. PCC 6803 was compared to wild type and different complementation strains. Fluorescence microscopy showed for the first time the in vivo kinetics of assembly and disassembly of the CP12-GapDH-PRK complex, which was absent in the mutant Δcp12. Metabolome analysis revealed differences in the contents of ribulose 1,5-bisphosphate and dihydroxyacetone phosphate, the products of the CP12-regulated enzymes GapDH and PRK, between wild type and mutant Δcp12 under changing CO2 conditions. Growth of Δcp12 was not affected at constant light under different inorganic carbon conditions, however, the addition of glucose inhibited growth in darkness as well as under diurnal conditions. The growth defect in the presence of glucose is associated with the inability of Δcp12 to utilize external glucose. These phenotypes could be complemented by ectopic expression of the native CP12 protein, however, expression of CP12 variants with missing redox-sensitive cysteine pairs only partly restored the growth with glucose. These experiments indicated that the loss of GapDH-inhibition via CP12 is more critical than PRK association. Measurements of the NAD(P)H oxidation revealed an impairment of light intensity-dependent redox state regulation in Δcp12. Collectively, our results indicate that CP12-dependent regulation of the CBC is crucial for metabolic adjustment under conditions leading to redox changes such as diurnal conditions, glucose addition, and different CO2 conditions in cyanobacteria.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

The social life of cyanobacteria.

The cyanobacterium Synechocystis secretes a specific sulphated polysaccharide to form floating cell aggregates.

Probing the biogenesis pathway and dynamics of thylakoid membranes.

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

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.

The Role of the Cyanobacterial Type IV Pilus Machinery in Finding and Maintaining a Favourable Environment.

Type IV pili (T4P) are proteinaceous filaments found on the cell surface of many prokaryotic organisms and convey twitching motility through their extension/retraction cycles, moving cells across surfaces. In cyanobacteria, twitching motility is the sole mode of motility properly characterised to date and is the means by which cells perform phototaxis, the movement towards and away from directional light sources. The wavelength and intensity of the light source determine the direction of movement and, sometimes in concert with nutrient conditions, act as signals for some cyanobacteria to form mucoid multicellular assemblages. Formation of such aggregates or flocs represents an acclimation strategy to unfavourable environmental conditions and stresses, such as harmful light conditions or predation. T4P are also involved in natural transformation by exogenous DNA, secretion processes, and in cellular adaptation and survival strategies, further cementing the role of cell surface appendages. In this way, cyanobacteria are finely tuned by external stimuli to either escape unfavourable environmental conditions via phototaxis, exchange genetic material, and to modify their surroundings to fit their needs by forming multicellular assemblies.

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria.

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane.

Membrane Dynamics in Phototrophic Bacteria.

Photosynthetic membranes are typically densely packed with proteins, and this is crucial for their function in efficient trapping of light energy. Despite being crowded with protein, the membranes are fluid systems in which proteins and smaller molecules can diffuse. Fluidity is also crucial for photosynthetic function, as it is essential for biogenesis, electron transport, and protein redistribution for functional regulation. All photosynthetic membranes seem to maintain a delicate balance between crowding, order, and fluidity. How does this work in phototrophic bacteria? In this review, we focus on two types of intensively studied bacterial photosynthetic membranes: the chromatophore membranes of purple bacteria and the thylakoid membranes of cyanobacteria. Both systems are distinct from the plasma membrane, and both have a distinctive protein composition that reflects their specialized roles. Chromatophores are formed from plasma membrane invaginations, while thylakoid membranes appear to be an independent intracellular membrane system. We discuss the techniques that can be applied to study the organization and dynamics of these membrane systems, including electron microscopy techniques, atomic force microscopy, and many variants of fluorescence microscopy. We go on to discuss the insights that havebeen acquired from these techniques, and the role of membrane dynamics in the physiology of photosynthetic membranes. Membrane dynamics on multiple timescales are crucial for membrane function, from electron transport on timescales of microseconds to milliseconds to regulation and biogenesis on timescales of minutes to hours. We emphasize the open questions that remain in the field.

Structural variability, coordination and adaptation of a native photosynthetic machinery.

Cyanobacterial thylakoid membranes represent the active sites for both photosynthetic and respiratory electron transport. We used high-resolution atomic force microscopy to visualize the native organization and interactions of photosynthetic complexes within the thylakoid membranes from the model cyanobacterium Synechococcus elongatus PCC 7942. The thylakoid membranes are heterogeneous and assemble photosynthetic complexes into functional domains to enhance their coordination and regulation. Under high light, the chlorophyll-binding proteins IsiA are strongly expressed and associate with Photosystem I (PSI), forming highly variable IsiA-PSI supercomplexes to increase the absorption cross-section of PSI. There are also tight interactions of PSI with Photosystem II (PSII), cytochrome b6f, ATP synthase and NAD(P)H dehydrogenase complexes. The organizational variability of these photosynthetic supercomplexes permits efficient linear and cyclic electron transport as well as bioenergetic regulation. Understanding the organizational landscape and environmental adaptation of cyanobacterial thylakoid membranes may help inform strategies for engineering efficient photosynthetic systems and photo-biofactories.

Loss of Filamentous Multicellularity in Cyanobacteria: the Extremophile Gloeocapsopsis sp. Strain UTEX B3054 Retained Multicellular Features at the Genomic and Behavioral Levels.

Multicellularity in Cyanobacteria played a key role in their habitat expansion, contributing to the Great Oxidation Event around 2.45 billion to 2.32 billion years ago. Evolutionary studies have indicated that some unicellular cyanobacteria emerged from multicellular ancestors, yet little is known about how the emergence of new unicellular morphotypes from multicellular ancestors occurred. Our results give new insights into the evolutionary reversion from which the Gloeocapsopsis lineage emerged. Flow cytometry and microscopy results revealed morphological plasticity involving the patterned formation of multicellular morphotypes sensitive to environmental stimuli. Genomic analyses unveiled the presence of multicellularity-associated genes in its genome. Calcein-fluorescence recovery after photobleaching (FRAP) experiments confirmed that Gloeocapsopsis sp. strain UTEX B3054 carries out cell-to-cell communication in multicellular morphotypes but at slower time scales than filamentous cyanobacteria. Although traditionally classified as unicellular, our results suggest that Gloeocapsopsis displays facultative multicellularity, a condition that may have conferred ecological advantages for thriving as an extremophile for more than 1.6 billion years.IMPORTANCECyanobacteria are among the few prokaryotes that evolved multicellularity. The early emergence of multicellularity in Cyanobacteria (2.5 billion years ago) entails that some unicellular cyanobacteria reverted from multicellular ancestors. We tested this evolutionary hypothesis by studying the unicellular strain Gloeocapsopsis sp. UTEX B3054 using flow cytometry, genomics, and cell-to-cell communication experiments. We demonstrate the existence of a well-defined patterned organization of cells in clusters during growth, which might change triggered by environmental stimuli. Moreover, we found genomic signatures of multicellularity in the Gloeocapsopsis genome, giving new insights into the evolutionary history of a cyanobacterial lineage that has thrived in extreme environments since the early Earth. The potential benefits in terms of resource acquisition and the ecological relevance of this transient behavior are discussed.

Factors Controlling Floc Formation and Structure in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Motile strains of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 readily aggregate into flocs, or floating multicellular assemblages, when grown in liquid culture. As described here, we used confocal imaging to probe the structure of these flocs, and we developed a quantitative assay for floc formation based on fluorescence imaging of 6-well plates. The flocs are formed from strands of linked cells, sometimes packed into dense clusters but also containing voids with very few cells. Cells within the dense clusters show signs of nutrient stress, as judged by the subcellular distribution of green fluorescent protein (GFP)-tagged Vipp1 protein. We analyzed the effects on flocculation of a series of mutations that alter piliation and motility, including Δhfq, ΔpilB1, ΔpilT1, and ΔushA mutations and deletion mutations affecting major and minor pilins. The extent of flocculation is increased in the hyperpiliated ΔpilT1 mutant, but active cycles of pilus extension and retraction are not required for flocculation. Deletion of PilA1, the major subunit of type IV pili, has no effect on flocculation; however, flocculation is lost in mutants lacking an operon coding for the minor pilins PilA9 to -11. Therefore, minor pilins appear crucial for flocculation. We show that flocculation is a tightly regulated process that is promoted by blue light perception by the cyanobacteriochrome Cph2. Floc formation also seems to be a highly cooperative process. A proportion of nonflocculating Δhfq cells can be incorporated into wild-type flocs, but the presence of a high proportion of Δhfq cells disrupts the large-scale architecture of the floc.IMPORTANCE Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation.

Using Nature's polyenes as templates: studies of synthetic xanthomonadin analogues and realising their potential as antioxidants.

Two truncated analogues of the polyenyl photoprotective xanthomonadin pigments have been synthesised utilising an iterative Heck-Mizoroki (HM)/iododeboronation cross coupling approach and investigated as models of the natural product photoprotective agents in bacteria. Despite the instability of these types of compounds, both analogues proved to be sufficiently stable to allow isolation, spectroscopic analysis and biological studies of their photoprotective behaviour which showed that despite their shorter polyene chain length, they retained the ability to protect bacteria from photochemical damage; i.e. incorporation of one compound into E. coli provided photoprotective activity against singlet oxygen analogous to the natural photoprotective mechanisms employed by Xanthomonas bacteria, answering key questions about what minimal functionality is required to impart photoprotection, potentially leading to new classes of photoprotective and antioxidants compounds.

Cyanobacterial Septal Junctions: Properties and Regulation.

Heterocyst-forming cyanobacteria are multicellular organisms that grow as chains of cells (filaments or trichomes) in which the cells exchange regulators and nutrients. In this article, we review the morphological, physiological and genetic data that have led to our current understanding of intercellular communication in these organisms. Intercellular molecular exchange appears to take place by simple diffusion through proteinaceous structures, known as septal junctions, which connect the adjacent cells in the filament and traverse the septal peptidoglycan through perforations known as nanopores. Proteins that are necessary to produce, and that may be components of, the septal junctions-SepJ, FraC and FraD-have been identified in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 model. Additionally, several proteins that are necessary to produce a normal number of nanopores and functional septal junctions have been identified, including AmiC-type amidases, peptidoglycan-binding proteins and some membrane transporters. Available reports and reevaluation of intercellular molecular transfer data for some mutants of Anabaena suggest that the septal junctions can be regulated, likely by a mechanism of gating.

How bacteria keep proteins moving.

Bacteria contain large numbers of negatively-charged proteins to avoid the electrostatic interactions with ribosomes that would dramatically reduce protein diffusion.

Light-controlled motility in prokaryotes and the problem of directional light perception.

The natural light environment is important to many prokaryotes. Most obviously, phototrophic prokaryotes need to acclimate their photosynthetic apparatus to the prevailing light conditions, and such acclimation is frequently complemented by motility to enable cells to relocate in search of more favorable illumination conditions. Non-phototrophic prokaryotes may also seek to avoid light at damaging intensities and wavelengths, and many prokaryotes with diverse lifestyles could potentially exploit light signals as a rich source of information about their surroundings and a cue for acclimation and behavior. Here we discuss our current understanding of the ways in which bacteria can perceive the intensity, wavelength and direction of illumination, and the signal transduction networks that link light perception to the control of motile behavior. We discuss the problems of light perception at the prokaryotic scale, and the challenge of directional light perception in small bacterial cells. We explain the peculiarities and the common features of light-controlled motility systems in prokaryotes as diverse as cyanobacteria, purple photosynthetic bacteria, chemoheterotrophic bacteria and haloarchaea.

Dissecting the Native Architecture and Dynamics of Cyanobacterial Photosynthetic Machinery.

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.

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.