Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

High-Light-Induced Stress Activates Lipid Deacylation at the Sn-2 Position in the Cyanobacterium Synechocystis Sp. PCC 6803.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) produce free fatty acids (FFAs) because the FFAs generated by deacylation of membrane lipids cannot be recycled. An engineered Aas-deficient mutant of Synechocystis sp. PCC 6803 grew normally under low-light (LL) conditions (50 µmol photons m-2 s-1) but was unable to sustain growth under high-light (HL) conditions (400 µmol photons m-2 s-1), revealing a crucial role of Aas in survival under the HL conditions. Several-times larger amounts of FFAs were produced by HL-exposed cultures than LL-grown cultures. Palmitic acid accounted for ∼85% of total FFAs in HL-exposed cultures, while C18 fatty acids (FAs) constituted ∼80% of the FFAs in LL-grown cultures. Since C16 FAs are esterified to the sn-2 position of lipids in the Synechocystis species, it was deduced that HL irradiation activated deacylation of lipids at the sn-2 position. Heterologous expression of FarB, the FFA exporter protein of Neisseria lactamica, prevented intracellular FFA accumulation and rescued the growth defect of the mutant under HL, indicating that intracellular FFA was the cause of growth inhibition. FarB expression also decreased the 'per-cell' yield of FFA under HL by 90% and decreased the proportion of palmitic acid to ∼15% of total FFA. These results indicated that the HL-induced lipid deacylation is triggered not by strong light per se but by HL-induced damage to the cells. It was deduced that there is a positive feedback loop between HL-induced damage and lipid deacylation, which is lethal unless FFA accumulation is prevented by Aas.

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the Cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PIIper se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

Production of glutamate and stereospecific flavors, (S)-linalool and (+)-valencene, by Synechocystis sp. PCC6803.

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.

A Novel Bacterial Nitrate Transporter Composed of Small Transmembrane Proteins.

A putative silent gene of the freshwater cyanobacterium Synechococcus elongatus strain PCC 7942, encoding a small protein with two transmembrane helices, was named nrtS, since its overexpression from an inducible promoter conferred nitrate uptake activity on the nitrate transport-less NA4 mutant of S. elongatus. Homologs of nrtS, encoding proteins of 67-118 amino acid residues, are present in a limited number of eubacteria including mostly cyanobacteria and proteobacteria, but some others, e.g. the actinobacteria of the Mycobacterium tuberculosis complex, also have the gene. When expressed in NA4, the nrtS homolog of the γ-proteobacterium Marinomonas mediterranea took up nitrate with higher affinity for the substrate as compared with the S. elongatus NrtS (Km of 0.49 mM vs. 2.5 mM). Among the 61 bacterial species carrying the nrtS homolog, the marine cyanobacterium Synechococcus sp. strain PCC 7002 is unique in having two nrtS genes (nrtS1 and nrtS2) located in tandem on the chromosome. Coexpression of the two genes in NA4 resulted in nitrate uptake with a Km (NO3-) of 0.15 mM, while expression of either of the two resulted in low-affinity nitrate uptake activity with Km values of >3 mM, indicating that NrtS1 and NrtS2 form a heteromeric transporter complex. The heteromeric transporter was shown to transport nitrite as well. A Synechococcus sp. strain PCC 7002 mutant defective in the nitrate transporter (NrtP) showed a residual activity of nitrate uptake, which was ascribed to the NrtS proteins. Blue-native PAGE and immunoblotting analysis suggested a hexameric structure for the NrtS proteins.

Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus.

BACKGROUND: Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required. RESULTS: Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L-1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L-1 h-1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L-1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems. CONCLUSIONS: Removal of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.

Modulation of the balance of fatty acid production and secretion is crucial for enhancement of growth and productivity of the engineered mutant of the cyanobacterium Synechococcus elongatus.

BACKGROUND: Among the three model cyanobacterial species that have been used for engineering a system for photosynthetic production of free fatty acids (FFAs), Synechococcus elongatus PCC7942 has been the least successful; the FFA-excreting mutants constructed from this strain could attain lower rates of FFA excretion and lower final FFA concentrations than the mutants constructed from Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002. It has been suggested that S. elongatus PCC7942 cells suffer from toxicity of FFA, but the cause of the low productivity has remained to be determined. RESULTS: By modulating the expression level of the acyl-acyl carrier protein thioesterase and raising the light intensity during cultivation, FFA secretion rates comparable to those obtained with the other cyanobacterial species were attained with an engineered Synechococcus elongatus mutant (dAS1T). The final FFA concentration in the external medium was also higher than previously reported for other S. elongatus mutants. However, about 85 % of the total FFA in the culture was found to remain in the cells, causing severe photoinhibition. Targeted inactivation of the wzt gene in dAS1T, which gene manipulation was previously shown to result in loss of the hydrophilic O-antigen layer on the cell surface, increased FFA secretion, alleviated photoinhibition, and lead to 50 and 45 % increase in the final cell density and the total amount of FFA in the culture (i.e., the sum of the cellular and extracellular FFA), respectively. The average rate of production of total FFA by the culture of the ∆wzt strain was 2.7 mg L(-1) h(-1), being five times higher than those reported for Synechocystis sp. PCC 6803 and comparable to the rates of triacylglycerol production in green algae. CONCLUSION: Synechococcus elongatus PCC7942 has larger capacity of FFA production than Synechocystis sp. PCC6803 but accumulates most of the product in the cell because of the imbalance of the rates of FFA production and secretion. This causes severe photoinhibition and exerts adverse effects on cell growth and FFA productivity. Enhancement of FFA secretion would be required to fully exploiting the capacity of FFA production for the purpose of biofuel production.

Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.

Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.

Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria.

Many of the cyanobacterial species found in marine and saline environments have a gene encoding a putative nitrite transporter of the formate/nitrite transporter (FNT) family. The presumed function of the gene (designated nitM) was confirmed by functional expression of the gene from the coastal marine species Synechococcus sp. strain PCC7002 in the nitrite-transport-less mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus strain PCC7942. The NitM-mediated nitrite uptake showed an apparent Km (NO2-) of about 8 µM and was not inhibited by nitrate, cyanate or formate. Of the nitM orthologs from the three oceanic cyanobacterial species, which are classified as α-cyanobacteria on the basis of the occurrence of Type 1a RuBisCO, the one from Synechococcus sp. strain CC9605 conferred nitrite uptake activity on NA4, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 did not. A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids. The C-terminal sequence was not conserved in the NitM proteins from ß-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002. Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

Loss of cytochrome cM stimulates cyanobacterial heterotrophic growth in the dark.

Although cyanobacteria are photoautotrophs, they have the capability for heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and the WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp) and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from the WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding Cyt c(M). The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of the WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of the WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of the WT. These results suggest a suppressive role(s) for Cyt cM in regulation of heterotrophic activity.

Nitrite transport activity of a novel HPP family protein conserved in cyanobacteria and chloroplasts.

Some cyanobacterial genomes encode an integral membrane protein of the HPP family, which exhibited nitrite transport activity when expressed in the nitrite transport-less NA4 mutant of the cyanobacterium Synechococcus elongatus strain PCC 7942. AT5G62720 and AT3G47980 were found to encode Arabidopsis homologs of the cyanobacterial protein. The product of AT5G62720 was localized to the chloroplast envelope membrane and was shown to confer nitrite uptake activity on the NA4 mutant when expressed with an N-terminally truncated transit peptide or as a fusion with the N-terminal region of the cyanobacterial HPP family protein. Kinetic analyses showed that the Arabidopsis protein has much higher affinity for nitrite (K(m) = 13 µM) than the cyanobacterial protein (K(m) = 150 µM). Illuminated chloroplasts isolated from the mutant lines of AT5G62720 showed much lower activity of nitrite uptake than the chloroplasts isolated from the wild-type Col-0 plants, while the chloroplasts of the mutants of AT1G68570 (AtNPF3.1), the gene previously reported to encode a plastid nitrite transporter AtNitr1, showed wild-type levels of nitrite uptake activity. AT3G47980 was expressed in roots but not in shoots. It has a putative transit peptide similar to that of AT5G62720 and its fusion with the N-terminal region of the cyanobacterial HPP protein showed low but significant activity of nitrite transport in the cyanobacterial cell. Transcription of AT5G62720 (AtNITR2;1) and AT3G47980 (AtNITR2;2) was stimulated by nitrate under the control of the NIN-like proteins, suggesting that the HPP proteins represent nitrate-inducible components of the nitrite transport system of plastids.

In vitro and in vivo analyses of the role of the carboxysomal ß-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate.

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 µmol mg(-1) protein min(-1)) was only 30 % of that observed for the wild-type carboxysomes (0.71 µmol mg(-1) protein min(-1)), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO2 in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO2 for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO2 by the cytoplasmically localized Rubisco by counteracting the action of the CO2 hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO2 fixation by the cyanobacterium at CO2 levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.

Effects of high CO2 on growth and metabolism of Arabidopsis seedlings during growth with a constantly limited supply of nitrogen.

Elevated CO2 has been reported to stimulate plant growth under nitrogen-sufficient conditions, but the effects of CO2 on growth in a constantly nitrogen-limited state, which is relevant to most natural habitats of plants, remain unclear. Here, we maintained Arabidopsis seedlings under such conditions by growing a mutant with reduced nitrate uptake activity on a medium containing nitrate as the sole nitrogen source. Under nitrogen-sufficient conditions (i.e. in the presence of ammonium), growth of shoots and roots of both the wild type (WT) and the mutant was increased approximately 2-fold by elevated CO2. Growth stimulation of shoots and roots by elevated CO2 was observed in the WT growing with nitrate as the sole nitrogen source, but in the mutant grown with nitrate, the high-CO2 conditions stimulated only the growth of roots. In the mutant, elevated CO2 caused well-known symptoms of nitrogen-starved plants, including decreased shoot/root ratio, reduced nitrate content and accumulation of anthocyanin, but also had an increased Chl content in the shoot, which was contradictory to the known effect of nitrogen depletion. A high-CO2-responsive change specific to the mutant was not observed in the levels of the major metabolites, although CO2 responses were observed in the WT and the mutant. These results indicated that elevated CO2 causes nitrogen limitation in the seedlings grown with a constantly limited supply of nitrogen, but the Chl content and the root biomass of the plant increase to enhance the activities of both photosynthesis and nitrogen uptake, while maintaining normal metabolism and response to high CO2.

Evaluation of the effects of P(II) deficiency and the toxicity of PipX on growth characteristics of the P(II)-Less mutant of the cyanobacterium Synechococcus elongatus.

Among the known functions of the P(II) protein (the glnB gene product) in the cyanobacterium Synechococcus elongatus, negative regulation of the activity of PipX, a transcriptional co-activator of the NtcA regulon, has been thought to be essential for cell viability, because all the P(II)-less mutants thus far constructed carry spontaneous mutations in pipX. PipX is thus deduced to be a toxic protein, but its toxicity has not been clearly defined because of the lack of P(II)-deficient mutants carrying wild-type pipX. In this study, we developed a method to construct a targeted P(II)-less mutant of S. elongatus without the pipX mutation and determined the contribution of PipX to the detrimental effects of P(II) deficiency. Growth defects of the mutant were severe under nitrogen-replete conditions, i.e. in the presence of ammonium, but were also apparent under nitrogen-limited conditions. Genetic analyses indicated that the growth impairment observed under the nitrogen-limited conditions is largely due to the toxicity of PipX. Some of the phenotypes observed under the nitrogen-replete conditions, including reduced pigmentation and death of most of the cells after transfer from nitrogen-limited conditions to nitrogen-replete conditions, were ascribed to the toxicity of PipX, but inactivation of pipX only partially rescued the growth defect observed in the presence of ammonium, indicating the presence of an as yet unknown P(II) function(s) required for normal growth. Effects of ammonium addition on the nitrite uptake activity of the glnB mutant revealed a new function for P(II) in regulation of the activity of the ABC-type cyanate/nitrite transporter.

CmpR is important for circadian phasing and cell growth.

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status.

MarR-type transcriptional regulator ChlR activates expression of tetrapyrrole biosynthesis genes in response to low-oxygen conditions in cyanobacteria.

Oxygen is required for three enzyme reactions in chlorophyll and bilin biosynthesis pathways: coproporphyrinogen III oxidase (HemF), heme oxygenase (HO1), and Mg-protoporphyrin IX monomethylester cyclase (ChlA(I)). The cyanobacterium Synechocystis sp. PCC 6803 has alternative enzymes, HemN, HO2, and ChlA(II), to supply chlorophyll/bilins even under low-oxygen environments. The three genes form an operon, chlA(II)-ho2-hemN, that is induced in response to low-oxygen conditions to bypass the oxygen-dependent reactions. Here we identified a transcriptional regulator for the induction of the operon in response to low-oxygen conditions. A pseudorevertant, Δho1R, was isolated from a HO1-lacking mutant Δho1 that is lethal under aerobic conditions. Δho1R grew well even under aerobic conditions. In Δho1R, HO2 that is induced only under low-oxygen conditions was anomalously expressed under aerobic conditions to complement the loss of HO1. A G-to-C transversion in sll1512 causing the amino acid change from aspartate 35 to histidine was identified as the relevant mutation by resequencing of the Δho1R genome. Sll1512 is a MarR-type transcriptional regulator. An sll1512-lacking mutant grew poorly under low-oxygen conditions with a remarked decrease in Chl content that would be caused by the suppressed induction of the chlA(II) and hemN genes in Chl biosynthesis under low-oxygen conditions. These results demonstrated that Sll1512 is an activator in response to low-oxygen environments and that the D35H variant becomes a constitutive activator. This hypothesis was supported by a gel shift assay showing that the Sll1512-D35H variant binds to the DNA fragment upstream of the operon. We propose to name sll1512 chlR.

Regulation of nitrate assimilation in cyanobacteria.

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

Nitrate assimilatory genes and their transcriptional regulation in a unicellular red alga Cyanidioschyzon merolae: genetic evidence for nitrite reduction by a sulfite reductase-like enzyme.

Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs, which is able to grow on ammonium, as well as nitrate as sole nitrogen source. Based on the complete genome sequence, proteins for nitrate utilization, nitrate transporter (NRT) and nitrate reductase (NR), were predicted to be encoded by the neighboring nuclear genes CMG018C and CMG019C, respectively, but no typical nitrite reductase (NiR) gene was found by similarity searches. On the other hand, two candidate genes for sulfite reductase (SiR) were found, one of which (CMG021C) is located next to the above-noted nitrate-related genes. Given that transcripts of CMG018C, CMG019C and CMG021C accumulate in nitrate-containing media, but are repressed by ammonium, and that SiR and NiR are structurally related enzymes, we hypothesized that the CMG021C gene product functions as an NiR in C. merolae. To test this hypothesis, we developed a method for targeted gene disruption in C. merolae. In support of our hypothesis, we found that a CMG021G null mutant in comparison with the parental strain showed decreased cell growth in nitrate-containing but not in ammonium-containing media. Furthermore, expression of CMG021C in the nirA mutant of a cyanobacterium, Leptolyngbya boryana (formerly Plectonema boryanum), could genetically complement the NiR defect. Immunofluorescent analysis indicated the localization of CMG021C in chloroplasts, and hence we propose an overall scheme for nitrate assimilation in C. merolae.