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.

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions.IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2 These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

Expression of Shewanella oneidensis MR-1 [FeFe]-hydrogenase genes in Anabaena sp. strain PCC 7120.

H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O(2) sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2).

A major facilitator superfamily protein, HepP, is involved in formation of the heterocyst envelope polysaccharide in the cyanobacterium Anabaena sp. strain PCC 7120.

Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O(2)) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox(-) phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox(-) mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the P(glnA) promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep.

Mutations in genes patA and patL of Anabaena sp. strain PCC 7120 result in similar phenotypes, and the proteins encoded by those genes may interact.

PatA resembles a response regulator protein with a defective DNA-binding domain, and PatL (All3305) is a pentapeptide repeat protein. A yeast two-hybrid library identified PatL as a protein with which PatA may interact. Heterocysts of patA and patL Anabaena sp. form nearly exclusively terminally in long filaments, further linking the genes.

Identification of ten Anabaena sp. genes that under aerobic conditions are required for growth on dinitrogen but not for growth on fixed nitrogen.

Heterocysts are specialized cells required for aerobic fixation of dinitrogen by certain filamentous cyanobacteria. Numerous genes involved in the differentiation and function of heterocysts in Anabaena sp. strain PCC 7120 have been identified by mutagenizing and screening for mutants that require fixed nitrogen for growth in the presence of oxygen. We have verified that 10 Anabaena sp. genes, all1338, all1591, alr1728, all3278, all3520, all3582, all3850, all4019, alr4311, and all4388, identified initially by transposon mutagenesis, are such genes by complementing or reconstructing the original mutation and by determining whether the mutant phenotype might be due to a polar effect of the transposon. Elucidation of the roles of these genes should enhance understanding of heterocyst biology.

Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

Cyanobacteria use sunlight and water to produce hydrogen gas (H2), which is potentially useful as a clean and renewable biofuel. Photobiological H2 arises primarily as an inevitable by-product of N2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N2. Most of the 49 variants examined were deficient in N2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N2 atmosphere significantly increased their in vivo rates of H2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H2 in an aerobic, nitrogen-containing environment.

The insertion sequences of Anabaena sp. strain PCC 7120 and their effects on its open reading frames.

Anabaena sp. strain PCC 7120, widely studied, has 145 annotated transposase genes that are part of transposable elements called insertion sequences (ISs). To determine the entirety of the ISs, we aligned transposase genes and their flanking regions; identified the ISs' possible terminal inverted repeats, usually flanked by direct repeats; and compared IS-interrupted sequences with homologous sequences. We thereby determined both ends of 87 ISs bearing 110 transposase genes in eight IS families (http://www-is.biotoul.fr/) and in a cluster of unclassified ISs, and of hitherto unknown miniature inverted-repeat transposable elements. Open reading frames were then identified to which ISs contributed and others--some encoding proteins of predictable function, including protein kinases, and restriction endonucleases--that were interrupted by ISs. Anabaena sp. ISs were often more closely related to exogenous than to other endogenous ISs, suggesting that numerous variant ISs were not degraded within PCC 7120 but transferred from without. This observation leads to the expectation that further sequencing projects will extend this and similar analyses. We also propose an adaptive role for poly(A) sequences in ISs.

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp. strain PCC 7120.

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P(hetC)-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P(hetR)-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120.

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF A) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120.

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.

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.

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120.

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.

Signal transduction genes required for heterocyst maturation in Anabaena sp. strain PCC 7120.

How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.

Is the periplasm continuous in filamentous multicellular cyanobacteria?

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which individual cells exchange nutrients and, presumably, regulatory molecules. Unknown mechanisms underlie this exchange. Classical electron microscopy shows that filamentous cyanobacteria bear a Gram-negative cell wall comprising a peptidoglycan layer and an outer membrane that are external to the cytoplasmic membrane, and that the outer membrane appears to be continuous along the filament of cells. This implies that the periplasmic space between the cytoplasmic and outer membranes might also be continuous. We propose that a continuous periplasm could constitute a communication conduit for the transfer of compounds, which is essential for the performance of these bacteria as multicellular organisms.

Mutations in four regulatory genes have interrelated effects on heterocyst maturation in Anabaena sp. strain PCC 7120.

Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins.

A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase-transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.

Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120.

Photoreduction of dinitrogen by heterocyst-forming cyanobacteria is of great importance ecologically and for subsistence rice agriculture. Their heterocysts must have a glycolipid envelope layer that limits the entry of oxygen if nitrogenase is to remain active to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Genes alr5354 (hglD), alr5355 (hglC) and alr5357 (hglB) of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, and hglE of Nostoc punctiforme are required for synthesis of heterocyst envelope glycolipids. Newly identified Fox- mutants bear transposons in nearby open reading frames (orfs) all5343, all5345-asr5349 and alr5351-alr5358. Complementation and other analysis provide evidence that at least orfs all5343 (or a co-transcribed gene), all5345, all5347, alr5348, asr5350-alr5353 and alr5356, but not asr5349, are also required for a Fox+ phenotype. Lipid and sequence analyses suggest that alr5351-alr5357 encode the enzymes that biosynthesize the glycolipid aglycones. Electron microscopy indicates a role of all5345 through all5347 in the normal deposition of the envelope glycolipids.

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer.

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.

Identification of cyanobacterial cell division genes by comparative and mutational analyses.

We performed comparative and mutational analyses to define more comprehensively the repertoire of genes involved in cyanobacterial cell division. Genes ftsE, ftsI, ftsQ, ftsW, and (previously recognized) ftsZ, minC, minD, minE and sulA were identified as homologues of cell division genes of Gram-negative and Gram-positive bacteria. Transposon mutagenesis of Synechococcus elongatus PCC 7942 identified five additional genes, cdv1, cdv2, cdv3, ftn6 and cikA, involved in cell division. cdv1 encodes a presumptive periplasmic peptidyl-prolyl cis-trans isomerase. cdv2 has similarity to ylmF which, like divIVA, lies within the Gram-positive bacterial ylm gene cluster whose members have functions associated with division. Conservation of other ylm genes in cyanobacteria suggests that cyanobacteria and Gram-positive bacteria share specific division proteins. Two ylm homologues are also found in algal and plant genomes. cdv3 has low but significant similarity to divIVA, suggesting that minE and cdv3 both mediate division-site determination in cyanobacteria. In contrast, Gram-positive bacteria lack minE, and (Gram-negative) proteobacteria lack divIVA. ftn6, of unknown function, and the circadian input kinase, cikA, are specific to cyanobacteria. In S. elongatus, unlike in other bacteria, FtsZ rings are formed at sites occupied by nucleoids. Thus, the division machinery of cyanobacteria differs in its composition and regulation from that of Gram-negative and Gram-positive bacteria.