Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis. Supported by the National Natural Sciences Foundation of China (Grant No. 30500108) and Hundred Talents Program of Chinese Academy of Sciences.     

Genetic inactivation of the psaB gene in Synechocystis sp. PCC 6803 disrupts assembly of photosystem I

The reaction center of photosystem (PS) I is comprised of a heterodimer of homologous polypeptides, PsaA and PsaB. In order to investigate the biogenesis of PS I, the psaB gene was inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis 6803. This mutation resulted in disruption of stable PS I assembly, but PS II assembled normally. Expression of the psaA gene was not affected by the mutation, but PsaA protein was not detected, indicating that stable PsaA homodimers did not form. The ability to inactivate psaB makes it a viable target for site-directed mutagenesis.     

Targeted deletion of psaJ from the cyanobacterium Synechocystis sp. PCC 6803 indicates structural interactions between the PsaJ and PsaF subunits of photosystem I

Photosystem I catalyzes the light-driven oxidation of plastocyanin or cytochrome c ₆ and the reduction of ferredoxin or flavodoxin. PsaJ is a 4.4 kDa hydrophobic subunit of photosystem I from cyanobacteria and chloroplasts. To investigate the function of PsaJ, we generated a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 in which the psaJ gene is replaced by a gene for chloramphenicol resistance. Deletion of psaJ led to a reduction in the steady state RNA level from psaF which is located upstream from psaJ. Immunoquantification using an anti-PsaF antibody revealed a significant decrease in the amount of PsaF in membranes of the mutant strain. Trimeric photosystem I complexes isolated from the mutant strain using n-dodecyl -D-maltoside lacked PsaJ, contained ca. 80% less PsaF, but maintained wild-type levels of other photosystem I subunits. In contrast, the photosystem I purified using Triton X-100 contained less than 2% PsaF when compared to the wild type, showing the more extractable nature of PsaF in PsaJ-less photosystem I in the presence of Triton X-100. PsaE was more accessible to removal by NaI in a mutant strain lacking PsaF and PsaJ than in the wild type. The presence of PsaF in photosystem I from the PsaJ-less strain did not alter the increased susceptibility of PsaE to removal by NaI. These results indicate an interaction between PsaJ and PsaF in the organization of the complex.     

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

Site-directed mutagenesis of the basic residues 321K to321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803

CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.     

Sequencing and modification of psbB,the gene encoding the CP-47 protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.     

Oxygen evolving membranes and particles from the transformable cyanobacterium Synechocystis sp. PCC6803

Membranes and PS II particles retaining high rates of O₂-evolving activity have been isolated from the transformable cyanobacterium, Synechocystis sp. PCC6803. Membranes from cells grown under red light exhibit rates of O₂-evolution ranging from 500–700 mole O₂/mg chl/h. PS II particles are prepared by a simple procedure involving DEAE column chromatography of detergent extracts obtained by simultaneous treatment of membranes with octylglucoside and dodecylmaltoside. The isolated PS II fraction is enriched in polypeptides immunologically cross-reactive with polypeptides present in core reaction center preparations of spinach, exhibits 77 K fluorescence emission maxima at 685 and 696 nm, but not emission and absorption due to phycobilines and is capable of rates of O₂-evolution exceeding 1000 mole O₂/mg chl/h.Abbreviations DM dodecyl--D-maltoside - OG octyl--D-glucoside     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803

In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major chlorophyll-binding protein, was present in reduced but significant amounts in the thylakoid. Herbicide binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.Abbreviations CP chlorophyll-binding protein - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - ferricyanide K₃Fe(CN)₆ - HEPES N-(2-hydroxyelthyl)piperazine-N-(2-hydroxypropane sulfonic acid) - MES 2-(N-morpholino)-ethanesulfonic acid - PCC Pasteur Culture Collection - PCR polymerase chain reaction - PS photosystem - Q_A first quinone acceptor in PSII - Q_B second quinone acceptor in PSII - Z redox-active tyrosine (Y161) in D1 serving as electron carrier between the Mn cluster and P680     

Characterization of a glucosylglycerol-phosphate-accumulating,salt-sensitive mutant of the cyanobacterium Synechocystis sp. strain PCC 6803

Salt-sensitive mutants of Synechocystis were obtained by random cartridge mutagenesis, and one mutant (mutant 4) was characterized in detail. The salt tolerance of mutant 4 was reduced to about 20% of that of the wild-type. This was caused by a defect in the biosynthetic pathway of the osmoprotective compound glucosylglycerol (GG). Salt-treated cells of mutant 4 accumulated the intermediate glucosylglycerol-phosphate (GG-P). Only low levels of phosphate-free GG were detected. The phosphorylated form of GG was not osmoprotective and seemed to be toxic. In vitro enzyme assays revealed that GG-P-phosphatase activity was completely absent in mutant 4, while GG-P-synthase remained unchanged. The integration site of the aphII cartridge in mutant 4 and the corresponding wild-type region was cloned and sequenced. Mutant 4 was complemented to salt resistance after transformation by the cloned wild-type region. The integration of the cartridge led to a deletion of about 1.1 kb of the chromosomal DNA. This affected two of the identified putative protein coding regions, orfII and stpA. The ORFII protein shows a high degree of similarity to the receiver domain of response regulator proteins. Related sequences were not found for StpA. We assume that in mutant 4, regulatory genes necessary for the process of salt adaptation in Synechocystis are impaired. Received: 12 January 1996 / Accepted: 28 May 1996     

Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.     

Functional analysis of the PsbP-like protein (sll1418) in Synechocystis sp. PCC 6803

A recent proteomic analysis of the thylakoid lumen of Arabidopsis thaliana revealed the presence of several PsbP-like proteins, and a homologue to this gene family was detected in the genome of the cyanobacterium Synechocystis sp. PCC 6803 (Schubert M, Petersson UA, Haas BJ, Funk C, Schröder WP, Kieselbach T (2002) J Biol Chem 277, 8354–8365). Using a peptide-directed antibody against this cyanobacterial PsbP-like protein (sll1418) we could show that it was localized in the thylakoid membrane and associated with Photosystem II. While salt washes did not remove the PsbP-like protein from the thylakoid membrane, it was partially lost during the detergent-based isolation of PSII membrane fractions. In total cell extracts this protein is present in the same amount as the extrinsic PsbO protein. We did not see any significant functional difference between the wild-type and a PsbP-like insertion mutant.     

Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis sp. PCC 6803 and mammalian glucose transporters

Synechocystis sp. PCC 6803 is capable of facultative photoheterotrophy with glucose as the sole carbon source. Eight mutants that were unable to take up glucose were transformed with plasmids from pooled gene banks of wild-type Synechocystis DNA prepared in an Escherichia coli vector that does not replicate in Synechocystis. One mutant (EG216) could be complemented with all gene banks to restore ability for photoheterotrophic growth. One of the gene banks was fractionated into single clones and plasmid DNA from each clone used to complement EG216. This yielded a 1.5 kb DNA fragment that was sequenced. It contained one complete open reading frame (gtr) whose putative gene product displayed high sequence conservation with the xylose transporter of E. coli and the mammalian glucose transporters. Further, the isolated gtr gene interrupted in vitro by a kanamycin resistance cassette could be used to construct mutants from wild-type Synechocystis sp. PCC 6803 that lacked a functional glucose transporter, thus confirming the identity of the gtr gene with the glucose transporter gene. This is the first prokaryotic glucose transporter known to share a sequence relationship with mammalian glucose transporters and the first sugar transporter from a cyanobacterium characterized at the sequence level.