Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium.

Biological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities. IMPORTANCE The function of phycobilisomes, the large antenna protein complexes in heterocysts has long been debated. This study provides direct evidence of the involvement of these proteins in supporting nitrogenase activity in Anabaena 33047, a heterocystous cyanobacterium that has an affinity for high light intensities. This strain was previously known to be recalcitrant to genetic manipulation and, hence, despite its many appealing traits, remained largely unexplored. We developed a genetic modification system for this strain and generated a ΔnblA mutant that exhibited resistance to phycobilisome degradation upon nitrogen starvation. Physiological characterization of the strain indicated that PBS degradation is not essential for acclimation to nitrogen deficiency and retention of PBS is advantageous for nitrogenase function.

Molecular organizations and function of iron-stress-induced-A protein family in Anabaena sp. PCC 7120.

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions, and surround photosystem I (PSI) trimer with a ring formation. A cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, it is unknown how the IsiAs are associated with PSI. Here we report on molecular organizations and function of the IsiAs in this cyanobacterium. A deletion mutant of the isiA1 gene was constructed, and the four types of thylakoids were prepared from the wild-type (WT) and ΔisiA1 cells under iron-replete (+Fe) and iron-deficient (-Fe) conditions. Immunoblotting analysis exhibits a clear expression of the IsiA1 in the WT-Fe. The PSI-IsiA1 supercomplex is found in the WT-Fe, and excitation-energy transfer from IsiA1 to PSI is verified by time-resolved fluorescence analyses. Instead of the IsiA1, both IsiA2 and IsiA3 are bound to PSI monomer in the ΔisiA1-Fe. These findings provide insights into multiple-expression system of the IsiA family in this cyanobacterium.

Double strand break (DSB) repair in Cyanobacteria: Understanding the process in an ancient organism.

Cyanobacterial species, Anabaena/Nostoc and Chroococcidiopsis are highly radio-resistant indicating the presence of a robust DNA repair system. However, unlike the establishment of multiple DNA repair pathways in the radio-resistant Deinococcus, research on DNA repair in cyanobacteria has lagged far behind. Being ancient organisms, it is likely that the DNA repair mechanisms have evolved from cyanobacteria to the modern day bacteria. This review focuses on identifying and collating information on the major DNA repair proteins in cyanobacteria including re-annotation of recR and ndk, using Anabaena/Nostoc sp. strain PCC7120 as a model organism. Unlike most other bacteria, the DNA repair genes of cyanobacteria are not clustered in operons. Though the functional characterisation of most DNA repair proteins is lacking in cyanobacteria, a bioinformatic approach using sequences of DNA repair proteins from Anabaena PCC7120, has helped identify the possible protein-protein interactions, and build probable pathways of double strand break (DSB) repair. The emerging picture can be used as a guide to discern the biochemical and physiological roles of the different DNA repair proteins in Anabaena or Synechocystis, which can be manipulated genetically and establish the different DNA repair pathways in cyanobacteria, and their evolution with time.

Function analysis of RNase E in the filamentous cyanobacterium Anabaena sp. PCC 7120.

RNase E is an endoribonuclease and plays a central role in RNA metabolism. Cyanobacteria, as ancient oxygen-producing photosynthetic bacteria, also contain RNase E homologues. Here, we introduced mutations into the S1 subdomain (F53A), the 5'-sensor subdomain (R160A), and the DNase I subdomain (D296A) according to the key activity sites of Escherichia coli RNase E. The results of degradation assays demonstrated that Asp296 is important to RNase E activity in Anabaena sp. PCC 7120 (hereafter PCC 7120). The docking model of RNase E in PCC 7120 (AnaRne) and RNA suggested a possible recognition mechanism of AnaRne to RNA. Moreover, overexpression of AnaRne and its N-terminal catalytic domain (AnaRneN) in vivo led to the abnormal cell division and inhibited the growth of PCC 7120. The quantitative analysis showed a significant decrease of ftsZ transcription in the case of overexpression of AnaRne or AnaRneN and ftsZ mRNA could be directly degraded by AnaRne through degradation assays in vitro, indicating that AnaRne was related to the expression of ftsZ and eventually affected cell division. In essence, our studies expand the understanding of the structural and functional evolutionary basis of RNase E and lay a foundation for further analysis of RNA metabolism in cyanobacteria.

The model cyanobacteria Anabaena sp. PCC 7120 possess an intact but partially degenerated gene cluster encoding gas vesicles.

BACKGROUND: Bacterial gas vesicles, composed of two major gas vesicle proteins and filled with gas, are a unique class of intracellular bubble-like nanostructures. They provide buoyancy for cells, and thus play an essential role in the growth and survival of aquatic and soil microbes. Moreover, the gas vesicle could be applied to multimodal and noninvasive biological imaging as a potential nanoscale contrast agent. To date, cylinder-shaped gas vesicles have been found in several strains of cyanobacteria. However, whether the functional gas vesicles could be produced in the model filamentous cyanobacteria Anabaena sp. PCC 7120 remains controversial. RESULTS: In this study, we found that an intact gvp gene cluster indeed exists in the model filamentous cyanobacteria Anabaena sp. PCC 7120. Real-time PCR assays showed that the gvpA gene is constitutively transcribed in vivo, and its expression level is upregulated at low light intensity and/or high growth temperature. Functional expression of this intact gvp gene cluster enables the recombinant Escherichia coli to gain the capability of floatation in the liquid medium, thanks to the assembly of irregular gas vesicles. Furthermore, crystal structure of GvpF in combination with enzymatic activity assays of GvpN suggested that these two auxiliary proteins of gas vesicle are structurally and enzymatically conserved, respectively. CONCLUSIONS: Our findings show that the laboratory strain of model filamentous cyanobacteria Anabaena sp. PCC 7120 possesses an intact but partially degenerated gas vesicle gene cluster, indicating that the natural isolate might be able to produce gas vesicles under some given environmental stimuli for better floatation.

Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.

Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

Both Enolase and the DEAD-Box RNA Helicase CrhB Can Form Complexes with RNase E in Anabaena sp. Strain PCC 7120.

At present, little is known about the RNA metabolism driven by the RNA degradosome in cyanobacteria. RNA helicase and enolase are the common components of the RNA degradosome in many bacteria. Here, we provide evidence that both enolase and the DEAD-box RNA helicase CrhB can interact with RNase E in Anabaena (Nostoc) sp. strain PCC 7120 (referred to here as PCC 7120). Furthermore, we found that the C-terminal domains of CrhB and AnaEno (enolase of PCC 7120) are required for the interaction, respectively. Moreover, their recognition motifs for AnaRne (RNase E of PCC 7120) turned out to be located in the N-terminal catalytic domain, which is obviously different from those identified previously in Proteobacteria We also demonstrated in enzyme activity assays that CrhB can induce AnaRne to degrade double-stranded RNA with a 5' tail. Furthermore, we investigated the localization of CrhB and AnaRne by green fluorescent protein (GFP) translation fusion in situ and found that they both localized in the center of the PCC 7120 cytoplasm. This localization pattern is also different from the membrane binding of RNase E and RhlB in Escherichia coli Together with the previous identification of polynucleotide phosphorylase (PNPase) in PCC 7120, our results show that there is an RNA degradosome-like complex with a different assembly mechanism in cyanobacteria.IMPORTANCE In all domains of life, RNA turnover is important for gene regulation and quality control. The process of RNA metabolism is regulated by many RNA-processing enzymes and assistant proteins, where these proteins usually exist as complexes. However, there is little known about the RNA metabolism, as well as about the RNA degradation complex. In the present study, we described an RNA degradosome-like complex in cyanobacteria and revealed an assembly mechanism different from that of E. coli Moreover, CrhB could help RNase E in Anabaena sp. strain PCC 7120 degrade double-stranded RNA with a 5' tail. In addition, CrhB and AnaRne have similar cytoplasm localizations, in contrast to the membrane localization in E. coli.

RNase II binds to RNase E and modulates its endoribonucleolytic activity in the cyanobacterium Anabaena PCC 7120.

In Escherichia coli, the endoribonuclease E (RNase E) can recruit several other ribonucleases and regulatory proteins via its noncatalytic domain to form an RNA degradosome that controls cellular RNA turnover. Similar RNA degradation complexes have been found in other bacteria; however, their compositions are varied among different bacterial species. In cyanobacteria, only the exoribonuclease PNPase was shown to bind to the noncatalytic domain of RNase E. Here, we showed that Alr1240, a member of the RNB family of exoribonucleases, could be co-isolated with RNase E from the lysate of the cyanobacterium Anabaena PCC 7120. Enzymatic analysis revealed that Alr1240 is an exoribonuclease II (RNase II), as it only degrades non-structured single-stranded RNA substrates. In contrast to known RNase E-interacting ribonucleases, which bind to the noncatalytic domain of RNase E, the Anabaena RNase II was shown to associate with the catalytic domain of RNase E. Using a strain in which RNase E and RNase II were tagged in situ with GFP and BFP, respectively, we showed that RNase E and RNase II form a compact complex in vivo by a fluorescence resonance energy transfer (FRET) assay. RNase E activity on several synthetic substrates was boosted in the presence of RNase II, suggesting that the activity of RNase E could be regulated by RNase II-RNase E interaction. To our knowledge, Anabaena RNase II is an unusual ribonuclease that interacts with the catalytic domain of RNase E, and it may represent a new type of RNA degradosome and a novel mechanism for regulating the activity of the RNA degradosome. As Anabaena RNase E interacts with RNase II and PNPase via different regions, it is very likely that the three ribonucleases form a large complex and cooperatively regulate RNA metabolism in the cell.

A polyketide synthase HglEA, but not HglE2, synthesizes heterocyst specific glycolipids in Anabaena sp. PCC 7120.

Heterocysts are the specialized cells for nitrogen fixation in some filamentous cyanobacteria. To protect the oxygen labile nitrogen fixing enzyme, nitrogenase, heterocysts keep their inner environment microoxic by developing layers of barrier on the outside of their outer membranes. Heterocyst specific glycolipids (Hgls) are constituents of the layer of barrier and amphipathic compounds, synthesized from a very long chain fatty alcohol as a hydrophobic tail and a sugar as a polar head. In the model heterocystous cyanobacterium Anabaena sp. PCC 7120, Hgls are made of fatty alcohol with 26 carbons and a glucose, linked by an ether bond in alpha configuration. The fatty alcohol is synthesized via reactions of a polyketide synthase, HglEA. In Anabaena sp. PCC 7120, another polyketide synthase HglE2 shared more than 50% identity in an amino acid sequence with HglEA and is expected to be involved in Hgls synthesis. However, no direct evidence has been reported. Here, we experimentally show that HglEA is the contributor of Hgls synthesis, and that HglE2 is not involved in the development of the heterocyst specific glycolipid layer.

Interprotein electron transfer biohybrid system for photocatalytic H2 production.

Worldwide there is a large research investment in developing solar fuel systems as clean and sustainable sources of energy. The fundamental mechanisms of natural photosynthesis can provide a source of inspiration for these studies. Photosynthetic reaction center (RC) proteins capture and convert light energy into chemical energy that is ultimately used to drive oxygenic water-splitting and carbon fixation. For the light energy to be used, the RC communicates with other donor/acceptor components via a sophisticated electron transfer scheme that includes electron transfer reactions between soluble and membrane bound proteins. Herein, we reengineer an inherent interprotein electron transfer pathway in a natural photosynthetic system to make it photocatalytic for aqueous H2 production. The native electron shuttle protein ferredoxin (Fd) is used as a scaffold for binding of a ruthenium photosensitizer and H2 catalytic function is imparted to its partner protein, ferredoxin-NADP+-reductase (FNR), by attachment of cobaloxime molecules. We find that this 2-protein biohybrid system produces H2 in aqueous solutions via light-induced interprotein electron transfer reactions (TON > 2500 H2/FNR), providing insight about using native protein-protein interactions as a method for fuel generation.

Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120.

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 µM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

Development of a New Biocontainment Strategy in Model Cyanobacterium Synechococcus Strains.

Recent synthetic biology efforts have raised biosafety concerns for possible release of engineered cyanobacteria into natural environments. To address the issues, we developed a controllable metal ion induced biocontainment system for two model cyanobacteria. First, six ion-inducible promoters were respectively evaluated in both Synechococcus elongatus PCC 7942 and the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, leading to the identification of an iron ion-repressed promoter PisiAB with low leakage and a reduction-fold of 5.4 and 7.9, respectively. Second, holin-endolysin and nuclease NucA systems were introduced, the inhibition rate of which against two Synechococcus strains varied from 61% to 86.4%. Third, two toxin/antitoxin modules were identified capable of inducing programmed suicide in both Synechococcus strains after induction. Furthermore, an escape experiment was conducted and the results showed that the system was able to achieve an escape frequency below the detection limit of 10-9 after 3 days' duration, demonstrating the strategy integrating iron ion-inducible promoter PisiAB and that toxin/antitoxin modules could be a useful tool for cyanobacterium biocontainment.

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.

Effect of growth temperature on biosynthesis and accumulation of carotenoids in cyanobacterium Anabaena sp. PCC 7120 under diazotrophic conditions.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) ß-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of ß-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of ß-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between ß-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Alr2321, a multiple stress inducible glyoxalase I of Anabaena sp. PCC7120 detoxifies methylglyoxal and reactive species oxygen.

Abiotic stresses enhance the cellular level of reactive oxygen species (ROS) which consequently leads to toxic methylglyoxal (MG) production. Glyoxalases (GlyI & GlyII) catalyze the conversion of toxic MG into non-toxic lactic acid but their properties and functions have been overlooked in cyanobacteria. This is the first attempt to conduct a genome-wide analysis of GlyI protein (PF00903) from Anabaena sp. PCC7120. Out of total nine GlyI domain possessing proteins, only three (Alr2321, Alr4469, All1022) harbour conserve His/Glu/His/Glu metal binding site at their homologous position and are deficient in conserved region specific for Zn2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni2+ dependent active GlyI with catalytic efficiency 11.7 × 106 M-1 s-1. It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni2+ followed by Co2+ > Mn2+ > Cu2+ and no activity with Zn2+. Moreover, the expression of alr2321 was found to be maximally up-regulated under heat (19 fold) followed by cadmium, desiccation, arsenic, salinity and UV-B stresses. BL21/pGEX-5X2-alr2321 showed improved growth under various abiotic stresses as compared to BL21/pGEX-5X2 by increased scavenging of intracellular MG and ROS levels. Taken together, these results suggest noteworthy links between intracellular MG and ROS, its detoxification by Alr2321, a member of GlyI family of Anabaena sp. PCC7120, in relation to abiotic stress.

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85.

ß-barrel-shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell-wall of Gram-negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane-localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N-terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline-rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.

Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP-galactose 4-epimerase HgdA.

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaenasp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here, we studied the function of the putative nucleoside-diphosphate-sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site-directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdAmutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP-tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP-galactose 4-epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst-specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC.

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.

Morphological and isotopic changes of heterocystous cyanobacteria in response to N2 partial pressure.

Earth's atmospheric composition has changed significantly over geologic time. Many redox active atmospheric constituents have left evidence of their presence, while inert constituents such as dinitrogen gas (N2 ) are more elusive. In this study, we examine two potential biological indicators of atmospheric N2 : the morphological and isotopic signatures of heterocystous cyanobacteria. Biological nitrogen fixation constitutes the primary source of fixed nitrogen to the global biosphere and is catalyzed by the oxygen-sensitive enzyme nitrogenase. To protect this enzyme, some filamentous cyanobacteria restrict nitrogen fixation to microoxic cells (heterocysts) while carrying out oxygenic photosynthesis in vegetative cells. Heterocysts terminally differentiate in a pattern that is maintained as the filaments grow, and nitrogen fixation imparts a measurable isotope effect, creating two biosignatures that have previously been interrogated under modern N2 partial pressure (pN2 ) conditions. Here, we examine the effect of variable pN2 on these biosignatures for two species of the filamentous cyanobacterium Anabaena. We provide the first in vivo estimate of the intrinsic isotope fractionation factor of Mo-nitrogenase (εfix  = -2.71 ± 0.09‰) and show that, with decreasing pN2 , the net nitrogen isotope fractionation decreases for both species, while the heterocyst spacing decreases for Anabaena cylindrica and remains unchanged for Anabaena variabilis. These results are consistent with the nitrogen fixation mechanisms available in the two species. Application of these quantifiable effects to the geologic record may lead to new paleobarometric measurements for pN2 , ultimately contributing to a better understanding of Earth's atmospheric evolution.

Molecular and biochemical characterization of All0580 as a methylglyoxal detoxifying glyoxalase II of Anabaena sp. PCC7120 that confers abiotic stress tolerance in E. coli.

Abiotic stresses enhance cellular reactive oxygen species (ROS) level which results in toxic methylglyoxal (MG) production. Glyoxalases catalyze the conversion of toxic MG into non-toxic lactic acid whose properties and function are still unknown in cyanobacteria. This is the first attempt to characterize All0580 from Anabaena sp. PCC7120 as GlyII using in silico and wet lab approaches. Data of functional complementation of E. coli GlyII mutant (ΔgloB), enzyme kinetics and ESI-MS analysis suggested that All0580 harbors distinctive GlyII activity. The catalytic efficiency of All0580 (3 × 106 M-1 s-1) is higher than Arabidopsis GlyII. AAS analysis revealed the presence of a binuclear Zn/Fe centre in All0580 active site. The qRT-PCR of the target gene revealed maximum up-regulation in salinity followed by drought, arsenic, heat, and UV-B stresses. BL21/pET-21a-all0580 showed 1.5 to 10 fold increased growth and up to 4 fold decreased intracellular MG level as compared to BL21/pET-21a cells under various abiotic stresses and MG. A 39% drop in ROS generation by BL21/pET-21a-all0580 under MG stress suggested its potential to manage MG toxicity. Above attributes suggest that the hypothetical protein All0580 is a novel GlyII of cyanobacteria which heterologously confers tolerance to multiple abiotic stresses in E. coli.