A proteolytic pathway coordinates cell division and heterocyst differentiation in the cyanobacterium Anabaena sp. PCC 7120.

The filamentous, multicellular cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a prokaryotic model for the study of cell differentiation and cell-cell interactions. Upon combined-nitrogen deprivation, Anabaena forms a particular cell type, heterocyst, for aerobic nitrogen fixation. Heterocysts are semiregularly spaced among vegetative cells. Heterocyst differentiation is coupled to cell division, but the underlying mechanism remains unclear. This mechanism could be mediated by the putative protease HetF, which is a divisome component and is necessary for heterocyst differentiation. In this study, by suppressor screening, we identified PatU3, as a negative regulator acting downstream of HetF for cell division and heterocyst development. The inactivation of patU3 restored the capacity of cell division and heterocyst differentiation in the ΔhetF mutant, and overexpression of patU3 inhibited both processes in the wild-type background. We demonstrated that PatU3 was a specific substrate of the protease activity of HetF. Consequently, PatU3 accumulated in the hetF-deficient mutant, which was responsible for the resultant mutant phenotype. The cleavage site of PatU3 by HetF was mapped after the Arg117 residue, whose mutation made PatU3 resistant to HetF processing, and mimicked the effect of hetF deletion. Our results provided evidence that HetF regulated cell division and heterocyst differentiation by controlling the inhibitory effects of PatU3. This proteolytic pathway constituted a mechanism for the coordination between cell division and differentiation in a prokaryotic model used for studies on developmental biology and multicellularity.

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120.

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N2 -fixing conditions are investigated. Using a label-free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.

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.

Soft X-Ray Imaging of Cellular Carbon and Nitrogen Distributions in Heterocystous Cyanobacteria.

Soft x-ray microscopy (SXM) is a minimally invasive technique for single-cell high-resolution imaging as well as the visualization of intracellular distributions of light elements such as carbon, nitrogen, and oxygen. We used SXM to observe photosynthesis and nitrogen fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120, which can form heterocysts during nitrogen starvation. Statistical and spectroscopic analyses from SXM images around the K-absorption edge of nitrogen revealed a significant difference in the carbon-to-nitrogen (C/N) ratio between vegetative cells and heterocysts. Application of this analysis to soft x-ray images of Anabaena sp. PCC 7120 revealed inhomogenous C/N ratios in the cells. Furthermore, soft x-ray tomography of Anabaena sp. PCC 7120 revealed differing cellular C/N ratios, indicating different carbon and nitrogen distributions between vegetative cells and heterocysts in three dimensions.

Translational and rotational manipulation of filamentous cells using optically driven microrobots.

Optical cell manipulation has become increasingly valuable in cell-based assays. In this paper, we demonstrate the translational and rotational manipulation of filamentous cells using multiple cooperative microrobots automatically driven by holographic optical tweezers. The photodamage of the cells due to direct irradiation of the laser beam can be effectively avoided. The proposed method will enable fruitful biomedical applications where precise cell manipulation and less photodamage are required.

Spatio-Temporal Gene Induction Systems in the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120.

In the last decade, much progress has been made in the photosynthetic production of valuable products using unicellular cyanobacteria. However, production of some products requires dark, anaerobic incubation, which prevents practical applications using these organisms. Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that is easy to manipulate genetically. Upon nitrogen step-down, this strain differentiates heterocysts that retain micro-oxic conditions for nitrogen fixation. We have developed gene regulation tools in this cyanobacterium. However, lack of a cell type-specific gene induction system has prevented A. 7120 from becoming a bona fide attractive host for photosynthetic production. We validated the usability of two transcriptional ON riboswitches that respond to theophylline or adenine. We then created a cell type-specific gene induction system by combining the riboswitches and promoters specific to either heterocysts or vegetative cells. We also created another cell type-specific gene induction system using small RNA that activates translation. Consequently, our study has expanded the toolbox for gene regulation in cyanobacteria and has enabled spatio-temporal gene induction in multicellular cyanobacteria.

A Comprehensively Curated Genome-Scale Two-Cell Model for the Heterocystous Cyanobacterium Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a nitrogen-fixing filamentous cyanobacterium. Under nitrogen-limiting conditions, a fraction of the vegetative cells in each filament terminally differentiate to nongrowing heterocysts. Heterocysts are metabolically and structurally specialized to enable O2-sensitive nitrogen fixation. The functionality of the filament, as an association of vegetative cells and heterocysts, is postulated to depend on metabolic exchange of electrons, carbon, and fixed nitrogen. In this study, we compile and evaluate a comprehensive curated stoichiometric model of this two-cell system, with the objective function based on the growth of the filament under diazotrophic conditions. The predicted growth rate under nitrogen-replete and -deplete conditions, as well as the effect of external carbon and nitrogen sources, was thereafter verified. Furthermore, the model was utilized to comprehensively evaluate the optimality of putative metabolic exchange reactions between heterocysts and vegetative cells. The model suggested that optimal growth requires at least four exchange metabolites. Several combinations of exchange metabolites resulted in predicted growth rates that are higher than growth rates achieved by only considering exchange of metabolites previously suggested in the literature. The curated model of the metabolic network of Anabaena sp. PCC 7120 enhances our ability to understand the metabolic organization of multicellular cyanobacteria and provides a platform for further study and engineering of their metabolism.

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

The trpE gene negatively regulates differentiation of heterocysts at the level of induction in Anabaena sp. strain PCC 7120.

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotated trpE genes in Anabaena sp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream of trpE bound a central regulator of differentiation, HetR, in vitro and was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotype in vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in the trpE mutant.

Anabaena cell ageing monitored with confocal fluorescence spectroscopy.

Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell.

Bayesian-based aberration correction and numerical diffraction for improved lensfree on-chip microscopy of biological specimens.

Lensfree on-chip microscopy is an emerging imaging technique that can be used to visualize and study biological specimens without the need for imaging lens systems. Important issues that can limit the performance of lensfree on-chip microscopy include interferometric aberrations, acquisition noise, and image reconstruction artifacts. In this study, we introduce a Bayesian-based method for performing aberration correction and numerical diffraction that accounts for all three of these issues to improve the effective numerical aperture (NA) and signal-to-noise ratio (SNR) of the reconstructed microscopic image. The proposed method was experimentally validated using the USAF resolution target as well as real waterborne Anabaena flos-aquae samples, demonstrating improvements in NA by ∼25% over the standard method, and improvements in SNR of 2.8 and 8.2 dB in the reconstructed image when compared to the reconstructed images produced using the standard method and a maximum likelihood estimation method, respectively.

Cell envelope components influencing filament length in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.

Is vanadium a biometal for boreal cyanolichens?

Molybdenum (Mo) nitrogenase has long been considered the predominant isoenzyme responsible for dinitrogen fixation worldwide. Recent findings have challenged the paradigm of Mo hegemony, and highlighted the role of alternative nitrogenases, such as the vanadium-nitrogenase. Here, we first characterized homeostasis of vanadium (V) along with other metals in situ in the dinitrogen fixing cyanolichen Peltigera aphthosa. These lichens were sampled in natural sites exposed to various levels of atmospheric metal deposition. These results were compared with laboratory experiments where Anabaena variabilis, which is also hosting the V-nitrogenase, and a relatively close relative of the lichen cyanobiont Nostoc, was subjected to various levels of V. We report here that V is preferentially allocated to cephalodia, specialized structures where dinitrogen fixation occurs in tri-membered lichens. This specific allocation is biologically controlled and tightly regulated. Vanadium homeostasis in lichen cephalodia exposed to various V concentrations is comparable to the one observed in Anabaena variabilis and other dinitrogen fixing organisms using V-nitrogenase. Overall, our findings support current hypotheses that V could be a more important factor in mediating nitrogen input in high latitude ecosystems than previously recognized. They invite the reassessment of current theoretical models linking metal dynamics and dinitrogen fixation in boreal and subarctic ecosystems.

A storage-based model of heterocyst commitment and patterning in cyanobacteria.

When deprived of fixed nitrogen (fN), certain filamentous cyanobacteria differentiate nitrogen-fixing heterocysts. There is a large and dynamic fraction of stored fN in cyanobacterial cells, but its role in directing heterocyst commitment has not been identified. We present an integrated computational model of fN transport, cellular growth, and heterocyst commitment for filamentous cyanobacteria. By including fN storage proportional to cell length, but without any explicit cell-cycle effect, we are able to recover a broad and late range of heterocyst commitment times and we observe a strong indirect cell-cycle effect. We propose that fN storage is an important component of heterocyst commitment and patterning in filamentous cyanobacteria. The model allows us to explore both initial and steady-state heterocyst patterns. The developmental model is hierarchical after initial commitment: our only source of stochasticity is observed growth rate variability. Explicit lateral inhibition allows us to examine ΔpatS, ΔhetN, and ΔpatN phenotypes. We find that ΔpatS leads to adjacent heterocysts of the same generation, while ΔhetN leads to adjacent heterocysts only of different generations. With a shortened inhibition range, heterocyst spacing distributions are similar to those in experimental ΔpatN systems. Step-down to non-zero external fN concentrations is also investigated.

Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120.

The fixation of atmospheric N(2) by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N(2) fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N(2) fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF(+) motif, a palindrome at or close to position -35 that seems essential for heterocyst-specific expression of certain genes.

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.

The Sakaguchi reaction product quenches phycobilisome fluorescence, allowing determination of the arginine concentration in cells of Anabaena strain PCC 7120.

The filamentous cyanobacterium Anabaena fixes nitrogen in specialized cells called heterocysts. The immediate product of fixation, ammonia, is known to be assimilated by addition to glutamate to make glutamine. How fixed nitrogen is transported along the filament to the 10 to 20 vegetative cells that separate heterocysts is unknown. N-fixing heterocysts accumulate an insoluble polymer containing aspartate and arginine at the cell poles. Lockau's group has proposed that the polymer is degraded at the poles to provide a mobile carrier, arginine, to the vegetative cells (R. Richter, M. Hejazi, R. Kraft, K. Ziegler, and W. Lockau, Eur. J. Biochem. 263:163-169, 1999). We wished to use the Sakaguchi reaction for arginine to determine the relative cellular concentration of arginine along the filament. At present, the methods for measuring absorption of the Sakaguchi reaction product at 520 nm are insufficiently sensitive for that purpose. However, that product quenches the fluorescence of phycobiliproteins, which we have adapted to a determination of arginine. Our results are consistent with the proposal that arginine is a principal nitrogen carrier from heterocysts to vegetative cells in Anabaena.

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120.

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

Fluorescence spectroscopy study of heterocyst differentiation in Anabaena PCC 7120 filaments.

Filamentous Anabaena PCC 7120 differentiates nitrogen-fixing specialized cells called heterocysts at regular intervals following removal of combined nitrogen from the medium. Phycobiliproteins are degraded during differentiation. Heterocyst differentiation was followed at the single cell level by using confocal fluorescence microscopy. The presence of an enhanced fluorescence emission peak from allophycocyanin (APC) indicates that the degradation of the phycobilisomes during nitrogen deprivation possibly initiates at the linker between APC and photosystem II in a bottom-to-top disassembly model. Furthermore, the fluorescence emission peak around 650 nm provides an advantageous marker to identify early candidates for differentiation.

Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy.

Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PS II activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PS II activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PS II photochemical yields [Formula: see text] Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PS II as judged by photochemical yields, but a strongly reduced PS II-associated antenna as judged by decreased F 0. The possible importance of the functional PS II in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called "superbright cells") were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.