mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

The developmental regulator PatD modulates assembly of the cell-division protein FtsZ in the cyanobacterium Anabaena sp. PCC 7120.

FtsZ is a tubulin-like GTPase that polymerizes to initiate the process of cell division in bacteria. Heterocysts are terminally differentiated cells of filamentous cyanobacteria that have lost the capacity for cell division and in which the ftsZ gene is downregulated. However, mechanisms of FtsZ regulation during heterocyst differentiation have been scarcely investigated. The patD gene is NtcA dependent and involved in the optimization of heterocyst frequency in Anabaena sp. PCC 7120. Here, we report that the inactivation of patD caused the formation of multiple FtsZ-rings in vegetative cells, cell enlargement, and the retention of peptidoglycan synthesis activity in heterocysts, whereas its ectopic expression resulted in aberrant FtsZ polymerization and cell division. PatD interacted with FtsZ, increased FtsZ precipitation in sedimentation assays, and promoted the formation of thick straight FtsZ bundles that differ from the toroidal aggregates formed by FtsZ alone. These results suggest that in the differentiating heterocysts, PatD interferes with the assembly of FtsZ. We propose that in Anabaena FtsZ is a bifunctional protein involved in both vegetative cell division and regulation of heterocyst differentiation. In the differentiating cells PatD-FtsZ interactions appear to set an FtsZ activity that is insufficient for cell division but optimal to foster differentiation.

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine.

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal α/ß propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, ß-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.

The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation.

Many filamentous cyanobacteria respond to the external cue of nitrogen scarcity by the differentiation of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. Heterocysts follow a spatial pattern along the filament of two heterocysts separated by ca. 10-15 vegetative cells performing oxygenic photosynthesis. HetR is a transcriptional regulator that directs heterocyst differentiation. In the model strain Anabaena sp. PCC 7120, the HetR protein was observed in various oligomeric forms in vivo, including a tetramer that peaked with maximal hetR expression during differentiation. Tetramers were not detected in a hetR point mutant incapable of differentiation, but were conspicuous in an over-differentiating strain lacking the PatS inhibitor. In differentiated filaments the HetR tetramer was restricted to heterocysts, being undetectable in vegetative cells. HetR co-purified with RNA polymerase from Anabaena mainly as a tetramer. In vitro, purified recombinant HetR was distributed between monomers, dimers, trimers and tetramers, and it was phosphorylated when incubated with (γ-(32)P)ATP. Phosphorylation and PatS hampered the accumulation of HetR tetramers and impaired HetR binding to DNA. In summary, tetrameric HetR appears to represent a functionally relevant form of HetR, whose abundance in the Anabaena filament could be negatively regulated by phosphorylation and by PatS.

Cyt c6-3: A New Isoform of Photosynthetic Cyt c6 Exclusive to Heterocyst-Forming Cyanobacteria.

All known cyanobacteria contain Cyt c6, a small soluble electron carrier protein whose main function is to transfer electrons from the Cyt b6f complex to PSI, although it is also involved in respiration. We have previously described a second isoform of this protein, the Cyt c6-like, whose function remains unknown. Here we describe a third isoform of Cyt c6 (here called Cytc6-3), which is only found in heterocyst-forming filamentous cyanobacteria. Cyt c6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between Cyt c6-3 and Cyt c6 related to the general protein folding, Cyt c6-3 presents differential electrostatic surface features as compared with Cyt c6, its expression is not copper dependent and has a low reactivity towards PSI. According to the different expression pattern, functional reactivity and structural properties, Cyt c6-3 has to play an as yet to be defined regulatory role related to heterocyst differentiation.

Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments.

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.IMPORTANCECircadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena, under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.

Transcription activation by NtcA in the absence of consensus NtcA-binding sites in an anabaena heterocyst differentiation gene promoter.

Heterocyst differentiation is orchestrated by the N control transcriptional regulator NtcA and the differentiation-specific factor HetR. In Anabaena sp. strain PCC 7120, the devBCA operon is expressed from two different promoters activated upon N stepdown. The distal devB promoter (transcription start point [TSP] located at position -704) represents a canonical class II NtcA-activated promoter, including a consensus NtcA-binding site centered 39.5 nucleotides upstream from the TSP. Transcription activation from a second TSP (-454) requires NtcA and is impaired in hetR mutants. In a wild-type background, three different DNA fragments, including both or each individual promoter, directed gfp expression localized mainly to proheterocysts and heterocysts. Expression was undetectable in an ntcA background and, for the fragment including the proximal promoter alone, also in a hetR background. In spite of the absence of consensus NtcA-binding sequences between the two TSPs, NtcA was shown to interact with this DNA region, and NtcA and its effector, 2-oxoglutarate, were necessary and sufficient for in vitro transcription from the -454 TSP. No HetR binding to the DNA or in vitro transcription from the proximal devB TSP promoted by HetR alone were detected. However, a moderate positive effect of HetR on NtcA binding to the DNA between the two devB TSPs was observed. The proximal devB promoter appears to represent a suboptimal NtcA-activated promoter for which HetR may act as a coactivator, with the physiological effect of restricting gene activation to conditions of prevalence of high NtcA and HetR levels, such as those taking place during heterocyst differentiation.

FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120.

Knowledge on the regulatory mechanisms controlling iron homeostasis in cyanobacteria is limited. In Anabaena sp. PCC 7120, the ferric uptake regulator FurA is a constitutive and essential protein whose expression is induced under iron deprivation. Our previous analyses have shown that this protein acts as a global transcriptional regulator, controlling the expression of several genes belonging to different functional categories, including schT, a gene coding for a TonB-dependent schizokinen transporter. In the present study we analysed the impact of FurA overexpression and iron availability on the transcriptional modulation of a broad range of Anabaena iron uptake, transport, storage and cellular iron utilization mechanisms, including enzymes involved in siderophore biosynthesis, TonB-dependent siderophore outer membrane transporters, siderophore periplasmic binding proteins, ABC inner membrane permeases, ferritin Dps family proteins, and enzymes involved in tetrapyrrole biosynthesis. By combining reverse transcription-PCR analyses, electrophoretic mobility shift assays and DNase I footprinting experiments, we defined a variety of novel direct iron-dependent transcriptional targets of this metalloregulator, including genes encoding at least five enzymes involved in the tetrapyrrole biosynthesis pathway. The results unravel the role of FurA as the master regulator of iron homeostasis in Anabaena sp. PCC 7120, providing new insights into the Fur regulons in cyanobacteria.

[Decision making in cannabis users]. / Toma de decisiones en consumidores de cannabis.

Several neuropsychological studies have shown that chronic cannabis users have cognitive impairments, including decision-making process. Therefore, this study aims to evaluate the process, through the somatic marker hypothesis in a sample of 41 cannabis users compared with a control group of equal size, and to analyze the influence of age, sex, education level, age of onset and amount of daily consumption. In order to do that, the software "Cartas" (similar to the Iowa Gambling Task), was used, implementing its two versions: normal and reverse. The results show significant differences between cannabis users and control group in the normal and reverse task execution. By block analysis, the control group obtained higher scores in the normal task execution, however, in the reverse task, the differences between groups are present in the initial task execution but not final task execution. None of the analyzed variables (age, sex ...) are significantly related to task performance. These results suggest the existence of alterations in the decision making process of consumers cannabis, which may relate to the difficulty in generating somatic markers, and not for insensitivity punishments insensitivity.

The Role of Mre Factors and Cell Division in Peptidoglycan Growth in the Multicellular Cyanobacterium Anabaena.

Bacteria in general serve two main tasks: cell growth and division. Both processes include peptidoglycan extension to allow cell expansion and to form the poles of the daughter cells, respectively. The cyanobacterium Anabaena forms filaments of communicated cells in which the outer membrane and the peptidoglycan sacculus, which is engrossed in the intercellular regions between contiguous cells, are continuous along the filament. During the growth of Anabaena, peptidoglycan incorporation was weak at the cell periphery. During cell division, midcell peptidoglycan incorporation matched the localization of the divisome, and incorporation persisted in the intercellular septa, even after the division was completed. MreB, MreC, and MreD were located throughout the cell periphery and, in contrast to other bacteria, also to the divisome all along midcell peptidoglycan growth. In Anabaena mutants bearing inactivated mreB, mreC, or mreD genes, which showed conspicuous alterations in the filament morphology, consecutive septal bands of peptidoglycan growth were frequently not parallel to each other and were irregularly spaced along the filament, reproducing the disposition of the Z-ring. Both lateral and septal growth was impaired in strains down-expressing Z-ring components, and MreB and MreD appeared to directly interact with some divisome components. We propose that, in Anabaena, association with the divisome is a way for localization of MreB, MreC, and MreD at the cell poles, where they regulate lateral, midcell, and septal peptidoglycan growth with the latter being involved in localization and maintenance of the intercellular septal-junction protein structures that mediate cell-cell communication along the filament. IMPORTANCE Peptidoglycan surrounds the bacterial cell, being essential for the determination of the bacterium-specific morphology and survival. Peptidoglycan growth has been thoroughly investigated in some model rod-shaped bacteria, and more recently some representatives with disparate morphologies became into focus, revealing that patterns of peptidoglycan growth are much more diverse than previously anticipated. Anabaena forms filaments of communicated cells exhibiting features of multicellular organisms, such as the production of morphogens and coupled circadian oscillations. Here, we showed that Anabaena presented a distinct pattern of peptidoglycan growth characterized by continuous incorporation of material at the polar intercellular regions, contributing to assembling and maintaining the protein complexes that expand the septal peptidoglycan mediating intercellular molecular exchange in the filament.

Specific role of the cyanobacterial PipX factor in the heterocysts of Anabaena sp. strain PCC 7120.

The PipX factor is a regulatory protein that seems to occur only in cyanobacteria. In the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120, open reading frame (ORF) asr0485, identified as the pipX gene, is expressed mainly under conditions of combined-nitrogen deprivation dependent on the global N regulator NtcA and the heterocyst-specific regulator HetR. Primer extension and 5' rapid amplification of cDNA ends (RACE) analyses detected three transcription start points corresponding to a canonical NtcA-activated promoter (to which direct binding of NtcA was observed), an NtcA- and HetR-dependent promoter, and a consensus-type promoter, the last with putative -35 and -10 determinants. Activation of pipX took place in cells differentiating into heterocysts at intermediate to late stages of the process. Accordingly, disruption of pipX led to impaired diazotrophic growth, reduced nitrogenase activity, and impaired activation of the nitrogenase structural genes. The nitrogenase activity of the mutant was low under oxic conditions, likely resulting from inefficient protection against oxygen. In line with this, the activation of the coxB2A2C2 and coxB3A3C3 operons, encoding heterocyst-specific terminal respiratory oxidases responsible for internal oxygen removal, was deficient in the pipX mutant. Therefore, the Anabaena PipX factor shows a spatiotemporal specificity contributing to normal heterocyst function, including full activation of the nitrogenase structural genes and genes of the nitrogenase-protective features of the heterocyst.

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.

[Decision-making in drug-dependent patients]. / Toma de decisiones en pacientes drogodependientes.

INTRODUCTION: The somatic marker hypothesis proposes that certain body signals guide decision-making processes in an adaptive direction. To see the influence of these markers on decision-making we used the Iowa Gambling Task, through which several studies have shown impaired decision-making in drug-dependent patients. OBJECTIVE: To assess the performance of a sample of drug-dependent patients in a task that is sensitive to the measurement of decision-making process, so as to see whether there are significant alterations, and to compare the performance of these patients with that of non-users (in the normal and inverted versions). METHOD: The sample consisted of 66 people (33 addicts and 33 control participants). We used a semi-structured interview on socio-demographic variables and two versions of the "Cartas" task, a computerised version of the Iowa Gambling Task. Result. Significant differences were found between the control and experimental groups in execution of the normal task, but not of the inverted version. In total, 75.76% of the drug-dependent patients showed impaired task performance, as against 24.24% who performed it correctly. DISCUSSION: The results indicate the presence of alterations in the decision-making processes of drug-dependent patients, who did not perform as well as the control group. This may be due to difficulty in generating somatic states according to possible future consequences (myopia about the future) in patients addicted to drugs.

[Art therapy and the promotion of child development in a hospitalization context]. / A arteterapia e a promoção do desenvolvimento infantil no contexto da hospitalização.

This study aimed to evaluate and compare the development of hospitalized children before and after art therapy interventions. Qualitative case studies were undertaken in this descriptive-exploratory research, based on the developmental evaluation of the children. The study participants were five children between seven and ten years old, in the Hospital of Tropical Illnesses (HDT) in the city of Goiânia, state of Goiás, Brazil, in 2006. Results showed that art therapy interventions efficiently promoted children's development. Art therapy is a resource for positively channeling the variables of hospitalized children's development and for neutralizing affective factors that naturally appear, as well as for exposing the child's healthier potentials, which sometimes receive little stimulus in the context of hospitalization.

Robust, coherent, and synchronized circadian clock-controlled oscillations along Anabaena filaments.

Circadian clocks display remarkable reliability despite significant stochasticity in biomolecular reactions. We study the dynamics of a circadian clock-controlled gene at the individual cell level in Anabaena sp. PCC 7120, a multicellular filamentous cyanobacterium. We found significant synchronization and spatial coherence along filaments, clock coupling due to cell-cell communication, and gating of the cell cycle. Furthermore, we observed low-amplitude circadian oscillatory transcription of kai genes encoding the post-transcriptional core oscillatory circuit and high-amplitude oscillations of rpaA coding for the master regulator transducing the core clock output. Transcriptional oscillations of rpaA suggest an additional level of regulation. A stochastic one-dimensional toy model of coupled clock cores and their phosphorylation states shows that demographic noise can seed stochastic oscillations outside the region where deterministic limit cycles with circadian periods occur. The model reproduces the observed spatio-temporal coherence along filaments and provides a robust description of coupled circadian clocks in a multicellular organism.

Interactions of PatA with the Divisome during Heterocyst Differentiation in Anabaena.

The Anabaena organismic unit is a filament of communicating cells. Under conditions of nitrogen scarcity, some cells along the filament differentiate into heterocysts, which are specialized in the fixation of atmospheric N2 and provide the vegetative cells with N2 fixation products. At a certain stage, the differentiation process becomes irreversible, so that even when nitrogen is replenished, no return to the vegetative cell state takes place, possibly as a consequence of loss of cell division capacity. Upon N-stepdown, midcell FtsZ-rings were detected in vegetative cells, but not in differentiating cells, and this was also the case for ZipN, an essential protein that participates in FtsZ tethering to the cytoplasmic membrane and divisome organization. Later, expression of ftsZ was arrested in mature heterocysts. PatA is a protein required for the differentiation of intercalary heterocysts in Anabaena The expression level of the patA gene was increased in differentiating cells, and a mutant strain lacking PatA exhibited enhanced FtsZ-rings. PatA was capable of direct interactions with ZipN and SepF, another essential component of the Anabaena Z-ring. Thus, PatA appears to promote inhibition of cell division in the differentiating cells, allowing progress of the differentiation process. PatA, which in mature heterocysts was detected at the cell poles, could interact also with SepJ, a protein involved in production of the septal junctions that provide cell-cell adhesion and intercellular communication in the filament, hinting at a further role of PatA in the formation or stability of the intercellular structures that are at the basis of the multicellular character of AnabaenaIMPORTANCEAnabaena is a cyanobacterial model that represents an ancient and simple form of biological multicellularity. The Anabaena organism is a filament of cohesive and communicating cells that can include cells specialized in different tasks. Thus, under conditions of nitrogen scarcity, certain cells of the filament differentiate into heterocysts, which fix atmospheric nitrogen and provide organic nitrogen to the rest of cells, which, in turn, provide heterocysts with organic carbon. Heterocyst differentiation involves extensive morphological, biochemical, and genetic changes, becoming irreversible at a certain stage. We studied the regulation during heterocyst differentiation of several essential components of the Anabaena cell division machinery and found that protein PatA, which is required for differentiation and is induced in differentiating cells, interacts with essential cell division factors and destabilizes the cell division complex. This suggests a mechanism for establishment of commitment to differentiation by inhibition of cell division.

Transcriptional regulation of development in heterocyst-forming cyanobacteria.

Filamentous, heterocyst-forming cyanobacteria are among the simplest multicellular systems in Nature. In the absence of combined nitrogen, the filaments consist of vegetative cells that fix CO2 through oxygenic photosynthesis and micro-oxic heterocysts specialized for the fixation of N2 in a proportion of about 10 to 1. The development of a heterocyst-containing filament involves differentiation of vegetative cells into heterocysts in a process that requires a distinct gene expression program. Two transcription factors are strictly required, NtcA and HetR. The CRP-family protein NtcA directly activates the expression of multiple genes during heterocyst differentiation - in some cases assisted by coactivators including HetR - and in mature heterocysts, whereas HetR is needed to build high NtcA levels in differentiating heterocysts and directly activates some particular genes. A few other regulators of gene expression participate at specific differentiation steps, and a specific transcription factor, CnfR, activates nif gene expression under the micro-oxic conditions of the heterocyst.

ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the filamentous cyanobacterium Anabaena.

The organismic unit of heterocyst-forming cyanobacteria is a filament of communicating cells connected by septal junctions, proteinaceous structures bridging the cytoplasms of contiguous cells. This distinct bacterial organization is preserved during cell division. In Anabaena, deletion of the zipN gene could not be segregated. We generated strain CSL109 that expresses zipN from a synthetic regulatable promoter. Under conditions of ZipN depletion, cells progressively enlarged, reflecting restricted cell division, and showed drastic morphological alterations including cell detachment from the filaments, to finish lysing. In contrast to the wild-type localization in midcell Z-rings, FtsZ was found in delocalized aggregates in strain CSL109. Consistently, the proportion of membrane-associated to soluble FtsZ in fractionated cell extracts was lower in CSL109. Bacterial two-hybrid analysis showed that ZipN interacts with FtsZ and other cell-division proteins including cytoplasmic Ftn6 and SepF, and polytopic FtsW, FtsX, FtsQ and FtsI. Additionally, ZipN interacted with the septal protein SepJ, and in CSL109 depletion of ZipN was concomitant with a progressive loss of septal specificity of SepJ. Thus, in Anabaena ZipN represents an essential FtsZ membrane tether and an organizer of the divisome, and it contributes to the conformation of septal structures for filament integrity and intercellular communication.

Transcription activation by NtcA and 2-oxoglutarate of three genes involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

In Anabaena sp. strain PCC 7120, differentiation of heterocysts takes place in response to the external cue of combined nitrogen deprivation, allowing the organism to fix atmospheric nitrogen in oxic environments. NtcA, a global transcriptional regulator of cyanobacteria, is required for activation of the expression of multiple genes involved in heterocyst differentiation, including key regulators that are specific to the process. We have set up a fully defined in vitro system, which includes the purified Anabaena RNA polymerase, and have studied the effects of NtcA and its signaling effector 2-oxoglutarate on RNA polymerase binding, open complex formation, and transcript production from promoters of the hetC, nrrA, and devB genes that are activated by NtcA at different stages of heterocyst differentiation. Both RNA polymerase and NtcA could specifically bind to the target DNA in the absence of any effector. 2-Oxoglutarate had a moderate positive effect on NtcA binding, and NtcA had a limited positive effect on RNA polymerase recruitment at the promoters. However, a stringent requirement of both NtcA and 2-oxoglutarate was observed for the detection of open complexes and transcript production at the three investigated promoters. These results support a key role for 2-oxoglutarate in transcription activation in the developing heterocyst.

Role of two NtcA-binding sites in the complex ntcA gene promoter of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N(2) in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.