Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Aluminum Effects on Uptake and Metabolism of Phosphorus by the Cyanobacterium Anabaena cylindrica

Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Preor post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AIPO₄ never exceeded 10% of the total phosphate concentration. The uptake of ³²P-phosphorus is not disturbed by aluminum either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica.     

Expression of an Adenylate Cyclase Gene of Anabaena cylindrica in the Cyanobacterium Anabaena sp. Strain PCC 7120

A transformant of Anabaena 7120 was made by introducing a plasmidthat includes an adenylate cyclase gene of Anabaena cylindrica.Expression of this gene was driven by the bacterial tac promoter.Transformants accumulate cAMP 170 fold higher than the concentrationin the parental strain. The transformation resulted in the fragmentationof filaments in both nitrogen-replete and nitrogen-free media.It was suggested that this fragmentation caused the inhibitionof growth under nitrogen-fixing conditions. (Received December 26, 1997; Accepted April 30, 1998)     

Effects of Aminooxyacetate and Aminoacetonitrile on Glycolate and Ammonia Release by the Cyanobacterium Anabaena cylindrica

Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH₄-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica.     

Positive Selection Scanning Reveals Decoupling of Enzymatic Activities of Carbamoyl Phosphate Synthetase in Helicobacter pylori

In an effort to detect factors which may be under positive selection, a survey for such genes in two pathogenic strains of Helicobacter pylori (J99 and 26695) was performed. Based on an analysis of synonymous and nonsynonymous substitutions, we identified 19 candidate genes under positive selection. A search for homologues with known crystallographic structures revealed Escherichia coli carbomoyl phosphate synthetase as a homologue of H. pylori carbamoyl phosphate synthetase. Carbamoyl phosphate synthetase as isolated from E. coli is a heterodimeric enzyme that possesses two different but coupled functionalities and is involved in the first committed step in the separate biosynthetic pathways for arginine and pyrimidine nucleotides. In this study, we provide evidence indicating that one of these functionalities appears to be under selective pressure. Reports from previously published site-directed mutagenesis studies point to a decoupling of amidotransferase and synthetase activities. Implications of these findings for a metabolic enzyme under positive selection are discussed in terms of the mechanisms of H. pylori pathogenesis. Received: 11 June 2001 / Accepted: 12 September 2001     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Activation of sulphate in Anabaena cylindrica

Summary Crude cell-free extracts of Anabaena cylindrica synthesized adenosine-5-phosphosulphate (AP³⁵S) and 3-phosphoadenosine-5-phosphosulphate (PAP³⁵S) from ³⁵SO₄ ^2- in the presence of Mg²⁺, ATP and inorganic pyrophosphatase. Maximum AP³⁵S and PAP³⁵S were produced at pH 7.15 and 8.05, respectively. APS kinase was detected in the supernatant of crude cell-free extracts by a spectrophotometric procedure. ATP-Sulphurylase had an absolute requirement for Mg²⁺ and less than 30% AP³⁵S was formed when Mg²⁺ was replaced by either Mn²⁺ or Co²⁺. Nucleotide triphosphates other than ATP and 2-deoxyATP were ineffective in this reaction. Maximum enzyme activity was observed at equimolar concentrations of Mg²⁺ and ATP and excess of either of these was inhibitory. Other nucleotide triphosphates, like GTP, UTP, CTP, TTP, ITP, or 2-deoxyATP also inhibited the enzyme activity. Inhibition by GTP was competitive with respect to ATP. ATP-sulphurylase activity was not affected by cysteine, methionine or glutathione.Abbreviations APS adenosine-5-phosphosulphate - PAPS 3-phosphoadenosine-5-phosphosulphate     

Response of Alkaline Phosphatases in the Cyanobacterium Anabaena sp. FACHB 709 to Inorganic Phosphate Starvation

Alkaline phosphatases (APases) play a crucial role in phosphorus (P) metabolism and regulation, but their physiological functions largely remain unclear in cyanobacteria. Here, we identified four putative APase genes, designated as phoA-709, phoD1-709, phoD2-709, and phoS-709, in the cyanobacterium Anabaena sp. FACHB 709, and investigated their response to inorganic phosphate (P_i) starvation. With the exception of phoD2-709, three other APase genes were expressed at a constant and relative low level in P_i-replete medium, whereas the expression of all four APase genes was elevated in response to P_i starvation but phoA-709 significantly. However, disruption of phoA-709 did not affect the total APase activity but caused the expressional up-regulation of phoD1-709 and phoS-709 under P_i-sufficient and P_i-limiting conditions. These suggest that, the four APases of Anabaena sp. FACHB 709 are involved in P metabolism and regulation, and PhoA-709 is the main, yet dispensable, APase.     

Hydrogen evolution by photobleached Anabaena cylindrica

We have studied the evolution of hydrogen by photobleached filaments of the heterocystous bluegreen alga Anabaena cylindrica. The photobleached cells became orange-yellow due to the heavy accumulation of carotenoids. We found that the yellow filaments produced much larger amounts of hydrogen than the normal, green ones, while the nitrogenase activity responsible for hydrogen evolution increased to a lesser extent. We suggest that a reversible hydrogenase activity induced in photobleached filaments is responsible for the excess amount of hydrogen. 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibits the hydrogen evolution of the yellow filaments which produce much more oxygen and fix less CO₂ than the green filaments. Therefore we consider the water to be a possible electron source for this hydrogenase. The low efficiency of light energy conversion (0.3%) in nitrogenase-catalyzed H₂ evolution (Laczkó, 1980 Z. Pflanzenphysiol. 100, 241–245) is increased to 1.5–2% by the appearance of the reversible hydrogenase activity.Abbreviations Chl chlorophyll - Car carotenoids - Phy phycocyanin - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - PSI photosystem I - PSII photosystem II     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

The Study of Carbamoyl Phosphate Synthetase 1 Deficiency Sheds Light on the Mechanism for Switching On/Off the Urea Cycle

Carbamoyl phosphate synthetase 1(CPS1) deficiency(CPS1D) is an inborn error of the urea cycle having autosomal(2q34) recessive inheritance that can cause hyperammonemia and neonatal death or mental retardation. We analyzed the effects on CPS1 activity, kinetic parameters and enzyme stability of missense mutations reported in patients with CPS1 deficiency that map in the 20-k Da C-terminal domain of the enzyme. This domain turns on or off the enzyme depending on whether the essential allosteric activator of CPS1, N-acetylL-glutamate(NAG), is bound or is not bound to it. To carry out the present studies, we exploited a novel system that allows the expression in vitro and the purification of human CPS1, thus permitting site-directed mutagenesis. These studies have clarified disease causation by individual mutations, identifying functionally important residues, and revealing that a number of mutations decrease the affinity of the enzyme for NAG. Patients with NAG affinity-decreasing mutations might benefit from NAG site saturation therapy with N-carbamyl-Lglutamate(a registered drug, the analog of NAG). Our results, together with additional present and prior site-directed mutagenesis data for other residues mapping in this domain, suggest an NAG-triggered conformational change in the b4-a4 loop of the C-terminal domain of this enzyme. This change might be an early event in the NAG activation process. Molecular dynamics simulations that were restrained according to the observed effects of the mutations are consistent with this hypothesis, providing further backing for this structurally plausible signaling mechanism by which NAG could trigger urea cycle activation via CPS1.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

Protective mechanisms in photosynthesis of Anabaena cylindrica

The role of O₂ photoreduction was studied in intact cells of normal and photobleached Anabaena cylindrica Lemm. strain PCC 7122. We found that O₂ photoreduction represents a protective mechanism against over-reduction of the photosyn-thetic electron transport chain only in normal Anabaena cells. This protective mechanism was not functioning in photobleached cells in spite of the increased rate of photosynthetic electron flow. A new electron acceptor, the induced reversible hydrogenase, is suggested to be operating in photobleached Anabaena cylindrica .     

Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica

Summary High levels of glutamine synthetase, detected using both a biosynthetic assay (P _i release from ATP) and a -glutamyl transferase assay, are present in aerobically grown N₂-fixing cultures of Anabaena cylindrica. The enzyme is soluble, has a pH optimum of 6.5–7.5, with a peak at 7.1–7.2 (biosynthetic activity) or 6.9 (transferase activity), and a temperature optimum at 30°C–40°C. Partially purified preparations are stable in air at 5°C for at least 3 days. Mg²⁺, Mn²⁺, Co²⁺ and Ca²⁺ support high rates of biosynthetic activity, Zn²⁺ is less effective and Cu²⁺ and Ba²⁺ are ineffective.Enzyme activity is regulated at several levels: possibly by repression and derepression of the enzyme in response to NH₄ ⁺ level; by variation in the Mn²⁺: ATP ratio with optimum activity at a 1:1 ratio; by feed-back inhibition which may be of a cumulative type. The consensus of the evidence suggests the absence of a covalent enzyme modification of the type found in E. coli. Glutamine synthetase levels are almost twice as high on a protein basis in the heterocysts as in the vegetative cells. Apparent K _m values for whole filaments for NH₄ ⁺ and glutamate in the biosynthetic reactions are 1 mM and 2 mM respectively.     

Geographical Segregation of the Neurotoxin-Producing Cyanobacterium Anabaena circinalis

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.     

Metabolic Activities of Isolated Spores of Anabaena cylindrica

A method for the isolation of spores of Anabaena cylindricais described, and results concerning the metabolic activitiesof isolated spores are presented. Isolated spores fixed carbondioxide in the light at a lower rate and evolved carbon dioxidein the dark at a higher rate than the corresponding whole filaments.Nitrogen-fixing ability in isolated spores could be detectedneither with the isotopic method nor with the new acetylenetechnique. Isolated spores were able to germinate at the samerate as spores within the original cell population when transferredinto fresh culture medium.     

Detection of Circulating Tumor Cells in Hepatocellular Carcinoma Using Antibodies against Asialoglycoprotein Receptor,Carbamoyl Phosphate Synthetase 1 and Pan-Cytokeratin

Background

Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs.

Methods

The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients.

Results

ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects.

Conclusions

Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.