Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Characterization of HetR protein turnover in Anabaena sp. PCC 7120

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied. Received: 24 June 1997 / Accepted: 5 December 1997     

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N₂. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N₂ gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

Heterocyst patterns without patterning proteins in cyanobacterial filaments

We have quantitatively modeled heterocyst differentiation after fixed nitrogen step-down in the filamentous cyanobacterium Anabaena sp. PCC 7120 without lateral inhibition due to the patterning proteins PatS or HetN. We use cell growth and division together with fixed-nitrogen dynamics and allow heterocysts to differentiate upon the local exhaustion of available fixed nitrogen. Slow transport of fixed nitrogen along a shared periplasmic space allows for fast growing cells to differentiate ahead of their neighbors. Cell-to-cell variability in growth rate determines the initial heterocyst pattern. Early release of fixed nitrogen from committed heterocysts allows a significant fraction of vegetative cells to be retained at later times. We recover the experimental heterocyst spacing distributions and cluster size distributions of Khudyakov and Golden [Khudyakov, I.Y., Golden, J.W., 2004. Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040-16045].     

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

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

Transcription of the extended <Emphasis Type="Italic">hyp</Emphasis>-operon in <Emphasis Type="Italic">Nostoc</Emphasis>sp. strain PCC 7120

Background  

The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N₂-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120.     

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability inAnabaena sp. strain PCC 7120

When deprived of combined nitrogen, aerobically-grown filaments ofAnabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus inAnabaena.     

Maintenance of heterocyst patterning in a filamentous cyanobacterium

In the absence of sufficient combined nitrogen, some filamentous cyanobacteria differentiate nitrogen-fixing heterocysts at approximately every 10th cell position. As cells between heterocysts grow and divide, this initial pattern is maintained by the differentiation of a single cell approximately midway between existing heterocysts. This paper introduces a mathematical model for the maintenance of the periodic pattern of heterocysts differentiated by Anabaena sp. strain PCC 7120 based on the current experimental knowledge of the system. The model equations describe a non-diffusing activator (HetR) and two inhibitors (PatS and HetN) that undergo diffusion in a growing one-dimensional domain. The inhibitors in this model have distinct diffusion rates and temporal expression patterns. These unique aspects of the model reflect recent experimental findings regarding the molecular interactions that regulate patterning in Anabaena. Output from the model is in good agreement with both the temporal and spatial characteristics of the pattern maintenance process observed experimentally.     

Hydrogen Photoproduction by Immobilized N2-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process

We have investigated two approaches to enhance and extend H₂ photoproduction yields in heterocystous, N₂-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO₂ supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H₂ production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N₂ by periodic treatments of air, or air plus CO₂. These treatments restored the photosynthetic activity of the cells and led to a high level of H₂ production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO₂) but not in the Anabaena PCC 7120 strain (for which H₂ yields did not change after air treatments). The highest H₂ yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO₂ under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O₂ from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.     

Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N₂. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13–15 and 16–18% under air and 1% CO₂-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5–11 and 9.7–13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H₂ productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H₂ production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H₂ productivity by outdoor cultures of cyanobacteria in high-light environments.

     

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

Summary It 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.     

in-silico analysis suggests alterations in the function of XisA protein as a possible mechanism of butachlor toxicity in the nitrogen fixing cyanobacterium Anabaena sp. PCC 7120

Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp. PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation.     

Enhanced Resistance to UV-B Radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by Repeated Exposure

In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N₂-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats.     

Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120

The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.