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PubMed1999

The global nitrogen regulator NtcA regulates transcription of the signal transducer PII (GlnB) and influences its phosphorylation level in response to nitrogen and carbon supplies in the Cyanobacterium synechococcus sp. strain PCC 7942.

The PII protein is encoded by a unique glnB gene in Synechococcus sp. strain PCC 7942. Its expression has been analyzed in the wild type and in NtcA-null mutant cells grown under different conditions of nitrogen and carbon supply. RNA-DNA hybridization experiments revealed the presence of one transcript species 680 nucleotides long, whatever the nutrient conditions tested. A second transcript species, 620 nucleotides long, absent in the NtcA null mutant, was observed in wild-type cells that were nitrogen starved for 2 h under both high and low CO2 and in the presence of nitrate under a high CO2 concentration. Primer extension analysis indicated that the two transcript species are generated from two tandem promoters, a sigma70 Escherichia coli-type promoter and an NtcA-dependent promoter, located 120 and 53 nucleotides, respectively, from the glnB initiation codon. The NtcA-dependent promoter is up-regulated under the conditions mentioned above, while the sigma70 E. coli-type promoter displays constitutive levels of transcripts in the NtcA null mutant and slightly different levels in the wild-type cells, depending on the nitrogen and carbon supplies. In general, a good correlation between the amounts of the two transcript species and that of the PII protein was observed, as revealed by immunodetection with specific antibodies. The phosphorylation level of PII in the wild type is inversely correlated with nitrogen availability and directly correlated with higher CO2 concentration. This regulation is correspondingly less stringent in the NtcA null mutant cells. In contrast, the dephosphorylation of PII is NtcA independent.

Bacterial Proteins↗
PubMed2000

The Synechococcus strain PCC 7942 glnN product (glutamine synthetase III) helps recovery from prolonged nitrogen chlorosis.

We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacterium Synechococcus strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of glnN is impaired in NtcA- and P(II)-deficient mutants. The only parameter which was negatively affected in the glnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.

Bacterial Proteins↗
PubMed2000

A new circadian class 2 gene, opcA, whose product is important for reductant production at night in Synechococcus elongatus PCC 7942.

Gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to S. elongatus promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which purF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in which purF phasing is altered revealed a mutant that carries an insertion in the opcA gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the purF promoter is not altered by opcA inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in S. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The opcA gene, expected to be cotranscribed with fbp and zwf, was shown to have its own class 2 promoter, whereas the fbp promoter was determined to be in class 1. Thus, opcA expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases.

Circadian Rhythm↗
PubMed2001

Unusual regulatory elements for iron deficiency induction of the idiA gene of Synechococcus elongatus PCC 7942.

Expression of a thylakoid membrane-associated protein called IdiA (iron-deficiency-induced protein A) is highly elevated and tightly regulated by iron limitation in Synechococcus elongatus PCC 6301 and PCC 7942. Although this protein is not essential for photosystem II (PSII) activity, it plays an important role in protecting the acceptor side of PSII against oxidative damage, especially under iron-limiting growth conditions, by an unknown mechanism. We defined the iron-responsive idiA promoter by using insertional inactivation mutagenesis and reporter gene assays. A 67-bp DNA region was sufficient for full iron deficiency-inducible idiA promoter activity. Within this fragment is a palindromic sequence 4 bp upstream of a putative -35 promoter element, which resembles the binding site of FNR/CAP-type helix-turn-helix transcription factors. The absence of this palindromic sequence or a 3-bp mutation in a putative -10 region eliminated promoter activity completely. A previously identified candidate for a positively acting transcription factor is the IdiB protein, whose gene lies immediately downstream of idiA. IdiB shows strong similarity to helix-turn-helix transcription factors of the FNR/CAP family. A His(6x)-tagged IdiB that was overexpressed in Escherichia coli bound to a 59-bp fragment of the idiA regulatory region that included the palindrome. Although the idiA promoter lacks a consensus binding site for the iron-sensing regulator Fur, we attempted to inactivate fur in order to investigate the potential role of this factor. The resulting merodiploid mutants showed constitutive partial derepression of IdiA expression under iron-sufficient growth conditions. We concluded that IdiB is a specific iron-responsive regulator of idiA and that Fur has an indirect role in influencing idiA expression.

Bacterial Proteins↗
PubMed2001

Novel form of ClpB/HSP100 protein in the cyanobacterium Synechococcus.

Synechococcus sp. strain PCC 7942 has a second clpB gene that encodes a 97-kDa protein with novel features. ClpBII is the first ClpB not induced by heat shock or other stresses; it is instead an essential, constitutive protein. ClpBII is unable to complement ClpBI function for acquired thermotolerance. No truncated ClpBII version is normally produced, unlike other bacterial forms, while ectopic synthesis of a putative truncated ClpBII dramatically decreased cell viability.

Adaptation, Biological↗
PubMed2001

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942.

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.

Chaperonin 60↗
PubMed2001

Functional elements of the strong psbAI promoter of Synechococcus elongatus PCC 7942.

The psbAI gene of the cyanobacterium Synechococcus elongatus PCC 7942 is one of three psbA genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of psbAI is downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the psbAI message, promoter activity decreases upon exposure to high light. Unlike the other psbA genes, additional sequences upstream of the psbAI -35 element are required for expression. Mutagenizing the atypical psbAI -10 element from TCTCCT to TATAAT increased the magnitude of expression from both psbAI::lacZ and psbAI::luxAB fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes rpoD2 and sigC, in both wild-type and -10-element mutagenized backgrounds, resulted in elevated psbAI::luxAB expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of the psbAI message binds one or more proteins from an S. elongatus extract. The corresponding region of psbAII efficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.

Bacterial Proteins↗
PubMed2002

nblS, a gene involved in controlling photosynthesis-related gene expression during high light and nutrient stress in Synechococcus elongatus PCC 7942.

The HliA protein of the cyanobacterium Synechococcus elongatus PCC 7942 is a small, thylakoid-associated protein that appears to play a role in photoprotection; its transcript rapidly accumulates in response to high-intensity light (HL) and the hli gene family is required for survival of cells in high light. In order to discover regulatory factors involved in HL acclimation in cyanobacteria, a screen was performed for chemically generated mutants unable to properly control expression of the hliA gene in response to HL. One such mutant was identified, and complementation analysis led to the identification of the affected gene, designated nblS. Based on its deduced protein sequence, NblS appears to be a membrane-bound, PAS-domain-bearing, sensor histidine kinase of two-component regulatory systems in bacteria. The nblS mutant was unable to properly control light intensity-mediated expression of several other photosynthesis-related genes, including all three psbA genes and the cpcBA genes. The mutant was also unable to control expression of the hliA and psbA genes in response to low-intensity blue/UV-A light, a response that may be related to the HL-mediated regulation of the genes. Additionally, in response to nutrient deprivation, the nblS mutant was unable to properly control accumulation of the nblA transcript and associated degradation of the light-harvesting phycobilisomes. The nblS mutant dies more rapidly than wild-type cells following exposure to HL or nutrient deprivation, likely due to its inability to properly acclimate to these stress conditions. Thus, the NblS protein is involved in the control of a number of processes critical for altering the photosynthetic apparatus in response to both HL and nutrient stress conditions.

Amino Acid Sequence↗
PubMed2003

ldpA encodes an iron-sulfur protein involved in light-dependent modulation of the circadian period in the cyanobacterium Synechococcus elongatus PCC 7942.

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.

Amino Acid Sequence↗
PubMed2003

Signal transduction protein P(II) is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the P(II) signaling protein play central roles in global nitrogen control in cyanobacteria. A dependence on P(II) for NtcA-regulated transcription, however, has not been observed. In the present investigation, we examined alterations in gene expression following nitrogen deprivation in Synechococcus elongatus strain PCC 7942 and specifically the roles of NtcA and P(II). Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo [(35)S]methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis. Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of S. elongatus failed to respond in P(II)- and NtcA-deficient mutants. One of the proteins whose synthesis was down-regulated in a P(II)- and NtcA-dependent manner was RbcS, the small subunit of RubisCO. Quantification of its mRNA revealed that the abundance of the rbcLS transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in P(II) and NtcA mutant cells. To investigate further the relationship between P(II) and NtcA, fusions of the promotorless luxAB reporter genes to the NtcA-regulated glnB gene were constructed and these constructs were used to transform wild-type cells and P(II)(-) and NtcA(-) mutants. Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a P(II)-independent manner. By contrast, NtcA-dependent activation of glnB expression following combined-nitrogen deprivation was impaired in the absence of P(II). Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the P(II) signal transduction protein.

Bacterial Proteins↗
PubMed2004

Light control of hliA transcription and transcript stability in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The high-light-inducible proteins (HLIPs) of cyanobacteria are polypeptides involved in protecting the cells from high-intensity light (HL). The hliA gene encoding the HLIP from Synechococcus elongatus strain PCC 7942 is expressed in response to HL or low-intensity blue or UV-A light. In this study, we explore via Northern analysis details of the transcriptional regulation and transcript stability of the hliA gene under various light conditions. Transcript levels of the hliA gene increased dramatically upon a shift to HL or UV-A light to similar levels, followed by a rapid decrease in UV-A light, but not in HL, consistent with blue/UV-A light involvement in early stages of HL-mediated expression. A 3-min pulse of low-intensity UV-A light was enough to trigger hliA mRNA accumulation, indicating that a blue/UV-A photoreceptor is involved in upregulation of the gene. Low-intensity red light was found to cause a slight, transient increase in transcript levels (raising the possibility of red-light photoreceptor involvement), while light of other qualities had no apparent effect. No evidence was found for wavelength-specific attenuation of hliA transcript levels induced by HL or UV-A light. Transcript decay was slowed somewhat in darkness, and when photosynthetic electron transport was inhibited by darkness or treatment with DCMU, there appeared a smaller mRNA species that may represent a decay intermediate that accumulates when mRNA decay is slowed. Evidence suggests that upregulation of hliA by light is primarily a transcriptional response but conditions that cause ribosomes to stall on the transcript (e.g., a shift to darkness) can help stabilize hliA mRNA and affect expression levels.

Bacterial Proteins↗
PubMed2004

A novel gene (narM) required for expression of nitrate reductase activity in the cyanobacterium Synechococcus elongatus strain PCC7942.

A new class of mutants deficient in nitrate assimilation was obtained from the cyanobacterium Synechococcus elongatus strain PCC7942 by means of random insertional mutagenesis. A 0.5-kb genomic region had been replaced by a kanamycin resistance gene cassette in the mutant, resulting in inactivation of two genes, one of which was homologous to the recently characterized cnaT gene of Anabaena sp. strain PCC7120 (J. E. Frías, A. Herrero, and E. Flores, J. Bacteriol. 185:5037-5044, 2003). While insertional mutation of the cnaT homolog did not affect expression of the nitrate assimilation operon or the activity of the nitrate assimilation enzymes in S. elongatus, inactivation of the other gene, designated narM, resulted in specific loss of the cellular nitrate reductase activity. The deduced NarM protein is a hydrophilic protein consisting of 161 amino acids. narM was expressed constitutively at a low level. The narM gene has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. In most of the cyanobacterial strains, narM is located downstream of narB, the structural gene of the cyanobacterial nitrate reductase, suggesting the functional link between the two genes. NarM is clearly not the structural component of the cyanobacterial nitrate reductase. The narM insertional mutant normally expressed narB, indicating that narM is not the transcriptional regulator of the structural gene of nitrate reductase. These results suggested that narM is required for either synthesis of the prosthetic group of nitrate reductase or assembly of the prosthetic groups to the NarB polypeptide to form functional nitrate reductase in cyanobacteria.

Amino Acid Sequence↗
PubMed2004

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942.

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.

Base Sequence↗
PubMed2004

Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation.

Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism. This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum by natural transformation with synthetic DNA constructs. Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented. These methods are ligation of DNA fragments with T4 DNA ligase, and megaprimer PCR.

Chlorobium↗
PubMed2001

Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus.

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55 degrees C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 mumol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 mumol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30-90 mumol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).

Cyanobacteria↗
PubMed2001

Expression of a ketolase gene mediates the synthesis of canthaxanthin in Synechococcus leading to tolerance against photoinhibition, pigment degradation and UV-B sensitivity of photosynthesis.

The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign beta-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous beta-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 mumol m-2 s-1) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 mumol m-2 s-1 and UV-B radiation of 6.8 W m-2. Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 mumol m-2 s-1, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

Bacterial Proteins↗
PubMed2001

CP43', the isiA gene product, functions as an excitation energy dissipator in the cyanobacterium Synechococcus sp. PCC 7942.

Under conditions of iron deficiency certain cyanobacteria induce a chlorophyll (Chl)-binding protein, CP43', which is encoded by the isiA gene. We have previously suggested that CP43' functions as a nonradiative dissipator of light energy. To further substantiate its functional role an isiA overexpression construct was introduced into the genome of a cyanobacterium Synechococcus sp. PCC 7942 (giving isiAoe cells). The presence of functional CP43' in isiAoe cells was confirmed by Western blot as well as by the presence of a characteristic blueshift of the red Chl a absorption peak and a notable increase in the 77 K fluorescence peak at 685 nm. Compared to wild-type cells isiAoe cells, with induced CP43', had both smaller functional antenna size and decreased yields of room temperature Chl fluorescence at various light irradiances. These observations strongly suggest that isiAoe cells, with induced CP43', have an increased capacity for dissipating light energy as heat. In agreement with this hypothesis isiAoe cells were also more resistant to photoinhibition of photosynthesis than wild-type cells. Based on these results we have further strengthened the hypothesis that CP43' functions as a nonradiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.

Bacterial Proteins↗