Expression Of ntcA Research Articles

Dynamics of Cylindrospermopsin Production and Toxin Gene Expression in <i>Aphanizomenon ovalisporum</i>

Aphanizomenon ovalisporum is a cylindrospermopsin (CYN)-producing cyanobacteria species that due to its increasing worldwide distribution has become an important health alarm in the last few years. Several clusters of genes involved in CYN production have been described in different CYN+ cyanobacteria genera, named aoa for Aphanizomenon and cyr for Cylindrospermopsis and others strains. The sequences of those genes are highly similar, but a rearrangement in gene order is also observed. The information on the control of CYN production by gene expression is still scarce, especially in Aphanizomenon. To obtain further information about the control of CYN production in A. ovalisporum, we have quantified the intra and extracellular CYN content, during nine days in BG11 batch cultures under optimal conditions. In parallel, the expression of four genes related to CYN synthesis, aoaA-C and cyrJ, has been analyzed by real time q-PCR. The results show a similar pattern of total CYN accumulation and gene expression. Most of the CYN is found intracellularly. Considering the high nitrogen content in the CYN molecule, we have explored if nitrogen assimilation could be related to CYN synthesis. We found inside the aoaA and aoaC sequences several putative binding domains for the global nitrogen regulator NtcA. The pattern of the ntcA expression along the culture is similar to that of CYN accumulation. Our data suggest that CYN production in A. ovalisporum seems to be controlled both by the expression of genesaoa and ntcA, this last one suggesting the influence of available nitrogen; however, other regulation mechanisms of CYN synthesis cannot be discarded.

Influence of coexisting spiramycin contaminant on the harm of Microcystis aeruginosa at different nitrogen levels.

The influence of nitrogen on the effects of the antibiotic contaminant spiramycin on Microcystis aeruginosa was studied through a 7-day exposure test. At current contamination levels of 0.5-100 mg L(-1) for nitrogen and 0.1-0.4 μg L(-1) for spiramycin, the two factors significantly interacted with each other (p<0.05) on the synthesis of chlorophyll-a and protein, the production and release of microcystins as well as the expression of ntcA gene and mcyB gene in M. aeruginosa. Nitrogen significantly affected the toxicity of spiramycin on algal growth (p<0.05) via regulation of protein synthesis. The photosynthesis system including chlorophyll-a, the psbA gene, and the rbcL gene participated in stress responses to spiramycin. Coexisting spiramycin contaminant increased the harm of M. aeruginosa by stimulating the production and release of MCs at a nitrogen level of 0.5 mg L(-1), but alleviated M. aeruginosa pollution at higher nitrogen levels of 5-100 mg L(-1) by inhibiting algal growth, the production of microcystins and the expression of ntcA and mcyB. The nitrogen-dependent effects of spiramycin should be considered in the control of M. aeruginosa bloom in the presence of spiramycin.

Hormesis effects of amoxicillin on growth and cellular biosynthesis of Microcystis aeruginosa at different nitrogen levels.

Coexisting antibiotic contaminants have potential to regulate cyanobacterial bloom, and the regulation is likely affected by nitrogen supply. A typical cyanobaterium Microcystis aeruginosa was cultured with 0.05-50mgL(-1) of nitrogen and exposed to 100-600ngL(-1) of amoxicillin for 7days. Algal growth was not significantly (p > 0.05) affected by amoxicillin at the lowest nitrogen level of 0.05mgL(-1), stimulated by 600ngL(-1) of amoxicillin at a moderate nitrogen level of 0.5mgL(-1) and enhanced by 100-600ngL(-1) of amoxicillin at higher nitrogen levels of 5-50mgL(-1). Amoxicillin affected chlorophyll-a, psbA gene, and rbcL gene in a similar manner as algal growth, suggesting a regulation of algal growth via the photosynthesis system. At each nitrogen level, synthesis of protein and polysaccharides as well as production and release of microcystins (MCs) increased in response to environmental stress caused by amoxicillin. Expression of ntcA and mcyB showed a positive correlation with the total content of MCs under exposure to amoxicillin at nitrogen levels of 0.05-50mgL(-1). Nitrogen and amoxicillin significantly (p < 0.05) interact with each other on the regulation of algal growth, synthesis of chlorophyll-a, production and release of MCs, and expression of ntcA and mcyB. The nitrogen-dependent stimulation effect of coexisting amoxicillin contaminant on M. aeruginosa bloom should be fully considered during the combined pollution control of M. aeruginosa and amoxicillin.

Distribution and expression of the cyanate acquisition potential among cyanobacterial populations in oligotrophic marine waters

We assessed the significance of cyanate utilization in marine primary productivity from the distribution of a dedicated transporter (encoded by cynABD) in different ocean environments. Several lines of evidence indicate that the cyanate utilization potential is associated mainly with surface populations of Prochlorococcus. Spatial and temporal dimensions of cynA, cynS, and ntcA expression by picocyanobacteria in the northern Red Sea supported our previous finding that cynA transcripts accumulate under more stringent N‐limiting conditions. At the same time, cyanate utilization appeared to be more complex than suggested in our earlier publication, as we showed that picocyanobacteria also express their cyanate utilization potential under conditions where labile organic N compounds, such as urea, accumulate. These include N‐sufficient transient conditions that result from nutrient upwelling during early mixing events in autumn as well as during spring bloom conditions that follow deep mixing events. Our finding that cynA occurrence is common in diverse marine environments suggests that cyanate utilization may be of a more fundamental importance to picophytoplankton productivity than previously considered.

FurA influences heterocyst differentiation in Anabaena sp. PCC 7120

In Anabaena sp. PCC 7120, FurA is a global transcriptional regulator whose expression is strongly induced by NtcA in proheterocysts and remains stably expressed in mature heterocysts. In the present study, overexpression of furA partially suppressed heterocyst differentiation by impairing morphogenesis at an early stage. Recombinant purified FurA specifically bound in vitro to the promoter regions of ntcA, while quantitative RT-PCR analyses indicated that furA overexpression strongly affected the transient increase of ntcA expression that occurs shortly after nitrogen step-down. Overall, the results suggest a connection between iron homeostasis and heterocyst differentiation via FurA, by modulating the expression of ntcA.

Mutagenesis of the cysteine residues in the transcription factor NtcA from Anabaena PCC 7120 and its effects on DNA binding in vitro

NtcA is a transcription factor found in a wide variety of cyanobacteria. It is a key component in the control of the nitrogen metabolism, and regulates genes involved in ammonia assimilation, heterocyst differentiation and nitrogen fixation. NtcA expression is subject to nitrogen control, but there is also evidence that the binding of NtcA to DNA can be regulated by a redox mechanism involving the two cysteine residues in the NtcA protein from Anabaena PCC 7120. In order to investigate this further, the two cysteine residues in NtcA were mutated into alanine to give four variants of the protein: wild-type NtcA, the point-mutated variants Cys157Ala and Cys164Ala, as well as the double mutant Cys157Ala/Cys164Ala. The binding of a DNA probe containing a palindromic NtcA-binding motif was investigated by gel mobility shift analysis under non-reducing and reducing conditions. The experiments show that the DNA binding in vitro is stronger in the presence of the reducing agent DTT than in its absence. However, this effect is not due to breaking of a disulfide bond between the cysteine residues, since the double mutant containing no cysteines was also affected by DTT. A molecular model of a monomer of NtcA, based on the homologous cAMP receptor protein structure, was created in order to locate the positions of the cysteine residues. The NtcA model suggested that the positions of the sulfur atoms are not compatible with formation of a bond between them.

Expression of cyanobacterial genes involved in heterocyst differentiation and dinitrogen fixation along a plant symbiosis development profile.

Members of the cyanobiont genus Nostoc, forming an endosymbiosis with members of the angiosperm genus Gunnera, undergo a number of characteristic phenotypic changes during the development of the symbiosis, the genetic background of which is largely unknown. Transcription patterns of genes related to heterocyst differentiation and dinitrogen fixation and corresponding protein profiles were examined, using reverse transcription-polymerase chain reaction and Western blots, along a developmental (apex to mature parts) sequence in Gunnera magellanica and G. manicata and under mimicked symbiotic conditions in a free-living Gunnera isolate (Nostoc strain 0102). The hetR gene was highly expressed and correlated positively with an increase in heterocyst frequency and with ntcA expression, whereas nifH expression was already high close to the growing apex and glnB (P(II)) expression decreased along the symbiotic profile. Although gene expression appeared to be regulated to a large extent in the same fashion as in free-living cyanobacteria, significant differences were apparent, such as the overexpression of both hetR and ntcA and the contrasting down-regulation of glnB, features indicating important regulatory differences between symbiotic and free-living cyanobacteria. The significance of these findings is discussed in a symbiotic context.

NITROGEN STRESS RESPONSE OF PROCHLOROCOCCUS STRAIN PCC 9511 (OXYPHOTOBACTERIA) INVOLVES CONTRASTING REGULATION OF ntcA AND amt11

The ntcA and amt1 genes (encoding a transcriptional activator of genes involved in nitrogen metabolism and a high affinity ammonium transporter respectively) from the axenic Prochlorococcus strain PCC 9511 were amplified, cloned, and sequenced. Cultures of this strain were exposed to nitrogen deprivation, and the expression of ntcA and amt1 was monitored along with cell density, photochemical quantum yield (as determined from Fv/Fm measurements), and cellular C:N ratios. Nitrogen deprivation led to arrested cell growth, reduced photochemical quantum yields, and increased cellular C:N ratios. Surprisingly, transcript accumulation patterns of ntcA and amt1 were not correlated. High levels of amt1 transcript were observed in both nitrogen‐replete and ‐deplete conditions. Expression of amt1 declined under severe deprivation of either phosphorus or nitrogen and showed a positive correlation with photochemical quantum yield. In contrast, ntcA expression was specifically enhanced in the absence of ammonium. High ntcA transcript levels were maintained over time and did not decline with low Fv/Fm values under severe N deprivation. Although ntcA transcript levels correlated somewhat with cellular C:N ratios, at the onset of nitrogen deprivation increases in ntcA mRNA levels occurred before increases in cellular C:N ratios. Constitutively high ammonium transporter expression together with low cellular ammonium requirements are likely to have important implications for the success of Prochlorococcus in the nitrogen‐poor oligotrophic waters where they are most abundant. The function of NtcA in Prochlorococcus strain PCC 9511 remains unclear.

Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development.

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.

Redox control of ntcA gene expression in Synechocystis sp. PCC 6803. Nitrogen availability and electron transport regulate the levels of the NtcA protein.

In this work we have studied the influence of the cellular redox status in the expression of the Synechocystis sp. PCC 6803 ntcA gene. Two different ntcA transcripts with different 5' ends were detected, depending on the different dark/light or nitrogen availability conditions. Accumulation of a 0.8-kb ntcA message was light and nitrogen dependent, whereas a longer 1.2-kb ntcA transcript was neither light nor nitrogen regulated. NtcA protein levels increased concomitantly with the accumulation of the 0.8-kb ntcA transcript. The light-dependent accumulation of the ntcA gene and the NtcA protein was sensitive to electron transport inhibitors. In addition, Glc-grown Synechocystis sp. cells showed a similar ntcA expression pattern in darkness to that observed under illumination. These data suggested that electron transport, and not light per se may regulate ntcA gene expression. Primer extension analysis, together with gel mobility-shift assays, demonstrated that in vitro, the Synechocystis sp. NtcA protein specifically bound to the putative promoter region from the light/nitrogen-dependent ntcA transcript but not to that from the constitutive 1.2-kb ntcA mRNA. Band-shift experiments carried out in the presence of thiol oxidizing/modifiying agents and different reducing/oxidizing conditions suggested that NtcA binding to its own promoter was under a thiol-dependent redox mechanism. Our results suggest that the cellular redox status plays a central role in the autoregulatory mechanism of the NtcA protein.

NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by direct interaction with two inactivating factors (IF7 and IF17). IF7 and IF17 are homologous polypeptides encoded by the gifA and gifB genes respectively. We investigated the transcriptional regulation of these genes. Expression of both genes is maximum in the presence of ammonium, when GS is inactivated. Nitrogen starvation attenuates the ammonium-mediated induction of gifA and gifB as well as the ammonium-mediated inactivation of GS. Putative binding sites for the transcription factor NtcA were identified at -7.5 and -30.5 bp upstream of gifB and gifA transcription start points respectively. Synechocystis NtcA protein binding to both promoters was demonstrated by gel electrophoresis mobility shift assays. Constitutive high expression levels of both genes were found in a Synechocystis NtcA non-segregated mutant (SNC1), which showed a fourfold reduction in the ntcA expression. These experiments indicate a repressive role for NtcA on the transcription of gifA and gifB genes. Our results demonstrate that NtcA plays a central role in GS regulation in cyanobacteria, stimulating transcription of the glnA gene (GS structural gene) and suppressing transcription of the GS inactivating factor genes gifA and gifB.

Expression and purification of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120.

The transcription factor NtcA from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli. In order to optimize the expression of NtcA, random silent mutations were introduced at the 5' end of the DNA encoding the protein. To get as high a yield of pure protein as possible, different strategies of expression as well as purification conditions were used. Under optimal expression conditions, a high-level expression clone of NtcA was coexpressed with GroEL-ES at 37 degrees C. A hexahistidine tag was added to the N-terminus of the protein in order to allow purification on an IMAC affinity column. Expression followed by one purification step using IMAC affinity chromatography gave a yield of 30-40 mg pure NtcA protein per liter of bacterial culture. Gel-shift experiments showed that the recombinant NtcA was active in binding a DNA sequence containing an NtcA-specific site.

Regulation of ntcA expression and nitrite uptake in the marine Synechococcus sp. strain WH 7803.

NtcA is a transcriptional activator involved in global nitrogen control in cyanobacteria. In the absence of ammonium it regulates the transcription of a series of genes encoding proteins required for the uptake and assimilation of alternative nitrogen sources (I. Luque, E. Flores, and A. Herrero, EMBO J. 13:2862-2869, 1994). ntcA, present in a single copy in the marine Synechococcus sp. strain WH 7803, was cloned and sequenced. The putative amino acid sequence shows a high degree of identity to NtcA from freshwater cyanobacteria in two functional domains. The expression of ntcA was negatively regulated by ammonium from a putative transcription start point located downstream of an NtcA consensus recognition sequence. Addition of either rifampin or ammonium led to a rapid decline in ntcA transcript levels with half-lives of less than 2 min in both cases. Nitrate-grown cells showed high ntcA transcript levels, as well as the capacity for active nitrite uptake. However, ammonium-grown cells showed low levels of the ntcA transcript and did not utilize nitrite. The addition of ammonium to nitrite uptake-active cells resulted in a gradual decline in the rate of uptake over a 24-h period. Active nitrite uptake was not induced in cells transferred to medium lacking a nitrogen source despite evidence of elevated expression of ntcA, indicating that ntcA expression is not sufficient for uptake capacity to develop. Nitrate and nitrite addition led to the development of nitrite uptake, whereas the addition of leucine did not. Furthermore, nitrite addition triggered the de novo protein synthesis required for uptake capacity to develop. These data suggest that nitrite and nitrate act as specific inducers for the synthesis of proteins required for nitrite uptake.

Cloning, sequencing, and regulation of the global nitrogen regulator gene ntcA in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K.

In cyanobacteria, ammonium represses expression of proteins involved in nitrogen fixation and assimilation. The global nitrogen regulator gene ntcA encodes a DNA-binding protein, NtcA, that is a transcriptional activator of genes subject to nitrogen control. We report the cloning and sequencing of the ntcA gene from a nitrogen-fixing unicellular cyanobacterium, Cyanothece sp. strain BH68K. The gene comprises 678 nucleotides, and the deduced NtcA protein contains 226 amino acids with a predicted molecular weight of 25,026. In addition, ntcA mRNA levels were measured in cells grown under different nitrogen regimes. Under nitrogen-fixing conditions, ntcA transcripts were weakly expressed. Furthermore, ntcA expression was diminished or inversely proportional to nifHDK expression. Conversely, ntcA expression increased in nitrate-grown cells, and a concentration-dependent increase was seen in ammonium-grown cells up to 1 mM NH4Cl. These results indicate that ntcA is involved more in nitrogen assimilation than in nitrogen fixation and also imply that the rhythmic expression of ntcA and nifHDK transcription may be under the control of a circadian clock.