Iris Maldener

The DevBCA exporter is essential for envelope formation in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120

The gene devA of the filamentous heterocyst‐forming cyanobacterium Anabaena sp. strain PCC 7120 encodes a protein with high similarity to ATP‐binding cassettes of ABC transporters. Mutant M7 defective in the devA gene is arrested in the development of heterocysts at an early stage and is not able to fix N2 under aerobic conditions. The devA gene is differentially expressed in heterocysts. To gain a better understanding of the structural components of this putative ABC transporter, we determined the complete nucleotide sequence of the entire gene cluster. The two additional genes, named devB and devC, encode proteins with similarities to membrane fusion proteins (DevB) of several ABC exporters and to membrane‐spanning proteins (DevC) of ABC transporters in general. Site‐directed mutations in each of the three genes resulted in identical phenotypes. Heterocyst‐specific glycolipids forming the laminated layer of the envelope were identified in lipid extracts of M7 and in the site‐directed mutants. However, transmission electron microscopy revealed unequivocally that the glycolipid layer is missing in mutant M7. Ultrastructural analysis also confirmed a developmental block at an early stage of differentiation. The results of this study suggest that the devBCA operon encodes an exporter of glycolipids or of an enzyme that is necessary for the formation of the laminated layer. The hypothesis is proposed that an intact envelope could be required for further heterocyst differentiation.

Septum-Localized Protein Required for Filament Integrity and Diazotrophy in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.

Alr0397 Is an Outer Membrane Transporter for the Siderophore Schizokinen in Anabaena sp. Strain PCC 7120

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

Calcium-dependent protease of the cyanobacterium Anabaena : molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

SummaryIt has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N2. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca2+-dependent protease activity, were not impaired in heterocyst formation and grew on N2 as sole nitrogen source.

Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

The morphogene AmiC2 is pivotal for multicellular development in the cyanobacterium Nostoc punctiforme

Filamentous cyanobacteria of the order Nostocales are primordial multicellular organisms, a property widely considered unique to eukaryotes. Their filaments are composed of hundreds of mutually dependent vegetative cells and regularly spaced N2‐fixing heterocysts, exchanging metabolites and signalling molecules. Furthermore, they may differentiate specialized spore‐like cells and motile filaments. However, the structural basis for cellular communication within the filament remained elusive. Here we present that mutation of a single gene, encoding cell wall amidase AmiC2, completely changes the morphology and abrogates cell differentiation and intercellular communication. Ultrastructural analysis revealed for the first time a contiguous peptidoglycan sacculus with individual cells connected by a single‐layered septal cross‐wall. The mutant forms irregular clusters of twisted cells connected by aberrant septa. Rapid intercellular molecule exchange takes place in wild‐type filaments, but is completely abolished in the mutant, and this blockage obstructs any cell differentiation, indicating a fundamental importance of intercellular communication for cell differentiation in Nostoc. AmiC2–GFP localizes in the cell wall with a focus in the cross walls of dividing cells, implying that AmiC2 processes the newly synthesized septum into a functional cell–cell communication structure during cell division. AmiC2 thus can be considered as a novel morphogene required for cell–cell communication, cellular development and multicellularity.

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 filaments 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.

The outer membrane of a heterocyst-forming cyanobacterium is a permeability barrier for uptake of metabolites that are exchanged between cells

The multicellular Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts, which exchange nutritional and regulatory compounds with the neighbour photosynthetic vegetative cells. The outer membrane of this bacterium is continuous along the filament defining a continuous periplasmic space. The Anabaena alr0075, alr2269 and alr4893 gene products were characterized as Omp85‐like proteins, which are generally involved in outer membrane protein biogenesis. Open reading frame alr2269 is the first gene of an operon that also carries genes for lipopolysaccharide lipid A biosynthesis including alr2270 (an lpxC homologue). Strains carrying inactivating alr2269 or alr2270 constructs showed enhanced sensitivity to erythromycin, SDS, lysozyme and proteinase K suggesting that they produce an outer membrane with increased permeability. These strains further exhibited increased uptake of sucrose, glutamate and, to a lesser extent, a few other amino acids. Increased uptake of the same metabolites was obtained by mechanical fragmentation of wild‐type Anabaena filaments. These results document that the outer membrane is a permeability barrier for metabolites such as sucrose and glutamate, which are subjected to intercellular exchange in the diazotrophic filament of heterocyst‐forming cyanobacteria.

NtcA-Dependent Expression of the devBCA Operon, Encoding a Heterocyst-Specific ATP-Binding Cassette Transporter in Anabaena spp.

The devBCA operon, encoding subunits of an ATP-binding cassette exporter, is essential for differentiation of N(2)-fixing heterocysts in Anabaena spp. Nitrogen deficiency-dependent transcription of the operon and the use of its transcriptional start point, located 762 (Anabaena variabilis strain ATCC 29413-FD) or 704 (Anabaena sp. strain PCC 7120) bp upstream of the translation start site, were found to require the global nitrogen transcriptional regulator NtcA. Furthermore, NtcA was shown to bind in vitro to the promoter of devBCA.

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.