Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Cells of free-living nitrogen-fixing Nostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia. were examined for the presence of ornithine carbamoyl transferase (OCT) by native-PAGE/in situ activity stain, and SDS-PAGE/Western immunoblots. Transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of OCT in the Nostoc cells. Moreover, the effects of photoautotrophic and dark heterotrophic growth metabolism on growth, nitrogenase activity and in vivo citrulline synthesis were investigated. PAGE in combination with in situ activity staining demonstrated an in vitro active OCT with a molecular weight of approximately 80 kDa. SDS-PAGE/Western immunoblots revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Pisum sativum L. cv. Alaska). Immunolocalization demonstrated that the OCT protein was located both in vegetative cells and heterocysts. Using the particle analysis of an image processor, the labeling associated with the photosynthetic vegetative cells was calculated to be 75.6 (± 5.5) gold particles μm^?2 compared with 62.0 (± 7.5) in the nitrogen-fixing heterocysts. Glucose and fructose stimulated both cyanobacterial growth and nitrogenase activity in light and darkness. Addition of exogenous ornithine decreased nitrogenase activity. In light grown cells, additions of glucose and fructose in combination with ornithine not only stimulated growth and nitrogenase activity but also in vivo citrulline synthesis, measured as ¹⁴CO₂-fixation into [¹⁴C]-citrulline. In darkness no stimulation was observed on in vivo citrulline synthesis. The substantial stimulation of nitrogenase activity by additions of external glucose and fructose, both in the light and in darkness, was not followed by a simultaneous stimulation of in vivo citrulline synthesis.     

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     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Immunological characterization of hydrogenases in the nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 73102

N₂-fixing Nostoc sp. strain PCC 73102 was examined for the presence of hydrogenases. Native-PAGE/immunoblots demonstrated that two proteins with molecular masses of approximately 200 kDa and 215 kDa are immunologically related to hydrogenases purified from Bradyrhizobium japonicum, Azotobacter vinelandii, Methanosarcina barkeri, and Thiocapsa roseopersicina. SDS-PAGE/immunoblots showed that one polypeptide, with a molecular mass of about 58 kDa, is immunologically related to the hydrogenases purified from all the microorganisms mentioned above. In addition, two polypeptides, with molecular masses of approximately 34 and 70 kDa, are immunologically related to the hydrogenases purified from T. roseopersicina and M. barkeri respectively. Immunogold/transmission electron microscopy showed that the hydrogenase proteins are present in both the heterocysts and the vegetative cells.     

Cloning and expression of a putative cyclodextrin glucosyltransferase from the symbiotically competent cyanobacterium Nostoc sp. PCC 9229

A polymerase chain reaction-based method was used to isolate a Nostoc sp. PCC 9229 cDNA from infected glands of Gunnera chilensis. The complete gene sequence was isolated from a genomic Nostoc sp. PCC 9229 library. Sequence analysis showed 84% amino acid similarity to a putative cyclodextrin glycosyltransferase from Nostoc sp. PCC 7120 and the gene was therefore termed cgt. Southern blot revealed that the cgt gene was present in symbiotically competent cyanobacteria. The cgt gene was expressed in free-living nitrogen-fixing cultures in light or in darkness when supplemented with fructose. This is the first expression analysis of a cgt gene from a cyanobacterium.     

Effects of nitrogen source on the synthesis of the UV-screening compound, scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102

The effects of nitrogen source (N(2), NO(3)(-) and NH(4)(+)) on scytonemin synthesis were investigated in the heterocystous cyanobacterium Nostoc punctiforme PCC 73102. With the required UVA radiation included, Nostoc synthesized three to seven times more scytonemin while fixing nitrogen than when utilizing nitrate or ammonium. A similar increase in scytonemin synthesis occurred when nitrate or ammonium became depleted by growth and Nostoc switched to diazotrophic metabolism with the differentiation of heterocysts. In addition, UVA-exposed cultures grown in medium with both NO(3)(-) and NH(4)(+) synthesized some scytonemin but synthesis increased when NH(4)(+) was depleted and growth had become dependent on NO(3)(-) reduction. Although the mechanism is unclear, these results suggest that the greater the restriction in nitrogen accessibility, the greater the production of scytonemin. Perhaps the entire response may be an interaction between this restriction and a resultant sensitivity to UV radiation that acts as a cue for determining the level of scytonemin synthesis. Scytonemin is a stable UVR screening compound and appears to be synthesized by cyanobacteria as a long-term solution for reducing UVR exposure and damage, but mainly or solely, when metabolic activity is absent. It is likely that during metabolic resurgence, the presence of a dense scytonemin sheath would facilitate the recovery process without the need for active defenses against UV radiation.     

Partial Purification and Characterization of Ornithine Carbamoyl Transferase (OCT) from the Cyanobacterium Nostoc sp. Strain PCC 73102

Ornithine carbamoyl transferase (OCT) catalyzes the formation of citrulline and orthophosphate from ornithine and carbamoyl phosphate. We have partially purified OCT from the filamentous cyanobacterium Nostoc sp. strain PCC 73102, using ammonium sulfate precipitation (35–55%), a gel-filtration column (Sephacryl S-200), followed by an affinity column (Sepharose-6B-PALO). The partially purified OCT was analyzed on native-PAGE and shown to be an active enzyme with an estimated molecular weight of approximately 80 kDa. The isoelectric point was determined to be about 6.2. Varying the ornithine concentration resulted in a hyperbolic response of the reaction velocity at lower concentrations. Ornithine concentrations above 2 mM inhibited the enzyme. A hyperbolic response of the OCT reaction was observed when increasing the carbamoyl phosphate concentration. From a double reciprocal plot, a saturation concentration of 0.8 mM and a V_max of 0.4 U/mg may be calculated. None of the tested compounds (argininosuccinate, arginine, aspartic acid, urea) had any significant positive effect on the in vitro activity of the partially purified OCT. Moreover, at concentrations higher than 10 mM, all tested compounds had an inhibitory effect. Received: 23 March 1998 / Accepted: 6 May 1998     

Isolation and characterization of a calmodulin-like protein from the cyanobacterium Nostoc sp. PCC 6720

A 21-kDa novel polypeptide which possesses characteristics normally considered to be diagnostic of the calmodulin present in eukaryotic cells was isolated from the cyanobacterium Nostoc sp. PCC 6720. The major technique employed in the isolation of the polypeptide was ion-exchange chromatography on a Mono Q column. The 21-kDa polypeptide was shown: to activate pea NAD kinase in vitro, in a Ca²⁺ requiring reaction; to react with polyclonal antibodies raised against spinach calmodulin, but not with those raised against bovine brain calmodulin; and to exhibit a Ca²⁺ dependent shift in migration during SDS-PAGE.Abbreviations ATCC American Type Culture Collection - DCPIP 2,6-dichlorophenylindophenol - PBS Phosphate buffered saline     

Relationship between color and pigment production in two stone biofilm-forming cyanobacteria (Nostoc sp. PCC 9104 and Nostoc sp. PCC 9025)

Previous studies have provided evidence that color measurements enable on site quantification of superficial biofilms, thereby avoiding the need for sampling. In the present study, the efficiency of color measurements to evaluate to what extent pigment production is affected by environmental parameters such as light intensity, combined nitrogen and nutrient availability, was tested with two cyanobacteria, Nostoc sp. strains PCC 9104 and PCC 9025, which form biofilms on stone. Both strains were acclimated, in aerated batch cultures for 2 weeks, to three different culture media: BG-11, BG-11₀, and BG-11₀/10 at either high or low light intensity. The content of chlorophyll a, carotenoids, and phycocyanins was measured throughout the experiment, together with variations in the color of the cyanobacteria, which were represented in the CIELAB color space. The results confirmed that the CIELAB color parameters are correlated with pigment content in such a way that variations in the latter are reflected as variations in color.     

Cloning,characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp. strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp. strain PCC7942. DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (M_{_r}, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a -type carbonic anhydrase (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression. Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO_{_2} to HCO^- _{_3} and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor. Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal -type CA.     

Cloning, Characterization, and Functional Expression in Escherichia coli of argH Encoding Argininosuccinate Lyase in the Cyanobacterium Nostoc sp. Strain PCC 73102

A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coliΔargH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea · min⁻¹· (mg protein)⁻¹. Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer. Received: 6 December 2000 / Accepted: 9 February 2001     

Role of mrgA in peroxide and light stress in the cyanobacterium Synechocystis sp. PCC 6803

In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the mrgA gene is part of the PerR regulon that is upregulated during peroxide stress. We determined that an Δ mrgA mutant was highly sensitive to low peroxide levels and that the mutant upregulated a gene cluster ( sll1722-26 ) that encoded enzymes involved with exopolymeric substance (EPS) production. We made mutants in this EPS cluster in both a wild type and Δ mrgA background and studied the responses to oxidative stress by measuring cell damage with LIVE/DEAD stain. We show that Synechocystis sp. PCC 6803 becomes highly sensitive to oxidative stress when either mrgA or the sll1722-26 EPS components are deleted. The results suggest that the deletion of the EPS cluster makes a cell highly susceptible to cell damage, under moderate oxidative stress conditions. Mutations in either mrgA or the EPS cluster also result in cells that are more light and peroxide sensitive, and produce significantly less EPS material than in wild type. In this study, we show that in the absence of MrgA, which is known to be involved in the storage or mobilization of iron, cells can be more easily damaged by exogenous oxidative and light stress.