Cluster of genes that encode positive and negative elements influencing filament length in a heterocyst-forming cyanobacterium.

The filamentous, heterocyst-forming cyanobacteria perform oxygenic photosynthesis in vegetative cells and nitrogen fixation in heterocysts, and their filaments can be hundreds of cells long. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the genes in the fraC-fraD-fraE operon are required for filament integrity mainly under conditions of nitrogen deprivation. The fraC operon transcript partially overlaps gene all2395, which lies in the opposite DNA strand and ends 1 bp beyond fraE. Gene all2395 produces transcripts of 1.35 kb (major transcript) and 2.2 kb (minor transcript) that overlap fraE and whose expression is dependent on the N-control transcription factor NtcA. Insertion of a gene cassette containing transcriptional terminators between fraE and all2395 prevented production of the antisense RNAs and resulted in an increased length of the cyanobacterial filaments. Deletion of all2395 resulted in a larger increase of filament length and in impaired growth, mainly under N2-fixing conditions and specifically on solid medium. We denote all2395 the fraF gene, which encodes a protein restricting filament length. A FraF-green fluorescent protein (GFP) fusion protein accumulated significantly in heterocysts. Similar to some heterocyst differentiation-related proteins such as HglK, HetL, and PatL, FraF is a pentapeptide repeat protein. We conclude that the fraC-fraD-fraE←fraF gene cluster (where the arrow indicates a change in orientation), in which cis antisense RNAs are produced, regulates morphology by encoding proteins that influence positively (FraC, FraD, FraE) or negatively (FraF) the length of the filament mainly under conditions of nitrogen deprivation. This gene cluster is often conserved in heterocyst-forming cyanobacteria.

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120.

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.

FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp.

The filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients and regulators. In Anabaena sp. strain PCC 7120, inactivation of sepJ or genes in the fraC operon (fraC, fraD and fraE) produce filament fragmentation. SepJ, FraC and FraD are cytoplasmic membrane proteins located in the filament's intercellular septa that are needed for intercellular exchange of the fluorescent tracer calcein (622 Da). Transmission electron microscopy showed an alteration in the heterocyst cytoplasmic membrane at the vegetative cell-heterocyst septa in ΔfraC and ΔfraD mutants. Immunogold labelling of FraD confirmed its localization in the intercellular septa and clearly showed the presence of part of the protein between the cytoplasmic membranes of the adjacent cells. This localization seemed to be affected in the ΔfraC mutant but was not impaired in a ΔsepJ mutant. Intercellular transfer of a smaller fluorescent tracer, 5-carboxyfluorescein (374 Da), was largely impaired in ΔfraC, ΔfraD and double ΔfraC-ΔfraD mutants, but much less in the ΔsepJ mutant. These results show the existence in the Anabaena filaments of a FraC/FraD-dependent intercellular molecular exchange that does not require SepJ.

Fra proteins influencing filament integrity, diazotrophy and localization of septal protein SepJ in the heterocyst-forming cyanobacterium Anabaena sp.

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 neighbouring photosynthetic vegetative cells. The cells in the filament appear to be joined by some protein structures, of which SepJ (FraG) that is located at the cell poles in the intercellular septa and is needed for filament integrity seems to be a component. Other known proteins required for filament integrity include FraC and FraH. Whereas fraC (alr2392) was constitutively expressed as an operon together with two downstream genes, alr2393 (fraD) and alr2394 (fraE), fraH (alr1603) was induced under nitrogen deprivation. Single mutants of these genes showed filament fragmentation under nitrogen deprivation and did not grow diazotrophically, although they formed heterocysts. The fraC and fraD mutants showed an impaired localization of SepJ at the intercellular septa and were hampered in the intercellular transfer of the fluorescent probe calcein. As shown with GFP fusions, FraC and FraD are also located at the intercellular septa. Therefore, at least three different proteins, SepJ, FraC and FraD, influence the architecture and function of the intercellular septa in the Anabaena filaments.

The amt gene cluster of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bears a gene cluster including three amt genes that, based on homology of their protein products, we designate amt4, amt1, and amtB. Expression of the three genes took place upon ammonium withdrawal in combined nitrogen-free medium and was NtcA dependent. The genes were transcribed independently, but an amt4-amt1 dicistronic transcript was also produced, and expression was highest for the amt1 gene. A mutant with the whole amt region removed could grow under laboratory conditions using ammonium, nitrate, or dinitrogen as the nitrogen source.