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Query: UMLS:C1832526 (PCC)
 5,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.
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PMID:Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties. 1516 46

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium Synechocystis sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the nrtABCD genes was completely inhibited by ammonium as in Synechococcus elongatus strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the Synechococcus strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the PII protein (glnB gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the Synechococcus strain. Mutants expressing the PII derivatives in which Ala or Glu is substituted for the conserved Ser49, which has been shown to be the phosphorylation site in the Synechococcus strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in Synechococcus either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the PII protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of PII, as was previously reported for Synechococcus elongatus strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.
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PMID:Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803. 1562 21

The phosphorylated signal transduction protein P(II) (P(II)-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of P(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired P(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15 % of total Mn(2+)-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual P(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of P(II) under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over P(II)-P, enabling the cells to rapidly dephosphorylate P(II)-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired P(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.
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PMID:Protein phosphatase PphA from Synechocystis sp. PCC 6803: the physiological framework of PII-P dephosphorylation. 1581 94

Summary In the filamentous cyanobacterium Anabaena sp. PCC 7120 patS and hetN suppress the differentiation of vegetative cells into nitrogen-fixing heterocysts to establish and maintain a pattern of single heterocysts separated by approximately 10 undifferentiated vegetative cells. Here we show that the patS- and hetN-dependent suppression pathways are the only major factors that prevent vegetative cells from differentiating into heterocysts when a source of ammonia is not present. The patS and hetN pathways are independent of each other, and inactivation of both patS and hetN leads to differentiation of almost all cells of a filament in the absence of a source of fixed nitrogen, compared with approximately 9% in the wild type. Complete differentiation of filaments also occurs when nitrate is supplied as a source of fixed nitrogen, conditions that do not induce differentiation of wild-type filaments. However, ammonia is still capable of suppressing differentiation. The percentage of cells that differentiate into heterocysts appears to be a function of time when a source of fixed nitrogen is absent or a function of growth phase when nitrate is supplied. Although differentiation proceeds unchecked in the absence of patS and hetN expression, differentiation is asynchronous and non-random.
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PMID:Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120. 1594 53

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.
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PMID:ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120. 1613 26

Signal transduction protein P(II) is dephosphorylated in Synechocystis sp. strain PCC 6803 by protein phosphatase PphA. To determine the impact of PphA-mediated P(II) dephosphorylation on physiology, the phenotype of a PphA-deficient mutant was analyzed. Mutants lacking either PphA or P(II) were impaired in efficient utilization of nitrate as the nitrogen source. Under conditions of limiting photosystem I (PSI)-reduced ferredoxin, excess reduction of nitrate along with impaired reduction of nitrite occurred in P(II) signaling mutants, resulting in excretion of nitrite to the medium. This effect could be reversed by increasing the level of PSI-reduced ferredoxin. We present evidence that nonphosphorylated P(II) controls the utilization of nitrate in response to low light intensity by tuning down nitrate uptake to meet the actual reduction capacity. This control mechanism can be bypassed by exposing cells to excess levels of nitrate. Uncontrolled nitrate uptake leads to light-dependent nitrite excretion even in wild-type cells, confirming that nitrate uptake controls nitrate utilization in response to limiting photon flux densities.
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PMID:Signal transduction protein PII phosphatase PphA is required for light-dependent control of nitrate utilization in synechocystis sp. strain PCC 6803. 1616 30

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.
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PMID:Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120. 1620 12

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Delta12 fatty acid desaturase mutant strains. Under a light intensity of 250 mumol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 20-38 degrees C, but growth was non-exponential below 20 degrees C and ceased at 12 degrees C. The Delta12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25-38 degrees C but grew slowly at 22 degrees C, and no cell growth took place below 18 degrees C. Under a very high-light intensity of 2.5 mmol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 30-38 degrees C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Delta12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 degrees C, but were killed at 30 degrees C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Delta12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 degrees C under the normal light intensity of 250 mumol m(-2) s(-1). These results suggest that growth of the Delta12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria.
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PMID:Synergistic effect of high-light and low temperature on cell growth of the Delta12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002. 1622 22

In this communication, we present a genetic analysis of the glnN promoter region of Synechococcus elongatus PCC 7942. luxAB reporter fusions were used to characterize the glnN promoter by deletion and site-directed mutational analysis. Reporter gene expression analysis was performed in S. elongatus wild-type and mutant strains to reveal the role of the global nitrogen responsive transcription factor NtcA and of the P(II) signalling protein on regulation of glnN gene expression. A non-canonical NtcA-binding motif is responsible for NtcA-dependent, nitrogen-responsive regulation of glnN. The P(II) signalling protein has opposing effects on NtcA-dependent glnN expression. Under conditions of nitrate-growth, it depresses expression, whereas under conditions of combined nitrogen starvation, it is required for full induction. Furthermore, sequences upstream of the NtcA-binding site have repressive effect on glnN promoter activity.
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PMID:Analysis of a non-canonical NtcA-dependent promoter in Synechococcus elongatus and its regulation by NtcA and PII. 1631 58

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr203, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.
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PMID:The nitrate/nitrite ABC transporter of Phormidium laminosum: phosphorylation state of NrtA is not involved in its substrate binding activity. 1644 36


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