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1.
Is There a Role for the 42 Kilodalton Polypeptide in Inorganic Carbon Uptake by Cyanobacteria? 总被引:8,自引:3,他引:5
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Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO2 conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (Ci), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO2 concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular Ci. The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular Ci pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of Ci, since the difference between the observed capabilities for CO2 and HCO3− uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO2-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO2 faster than the mutant. An analysis of the curves relating the rate of accumulation of Ci to the concentration of CO2 or HCO3− supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO2 was the preferred Ci species taken up by Synechococcus. 相似文献
2.
Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO(2)-Requiring Phenotype : Evidence for a Central Role for Carboxysomes in the CO(2) Concentrating Mechanism 总被引:10,自引:5,他引:5
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Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (Ci) and low Ci grown cells leads the creation of a high Ci requiring phenotype causing: (a) a dramatic increase in the K0.5 (Ci) for photosynthesis, (b) a loss of the ability to accumulate internal Ci, and (c) a decrease in the lag between the initial Ci accumulation following illumination and the efflux of CO2 from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO2 concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and Ci accumulation is developed in which the carboxysome plays a central role as both the site of CO2 generation from HCO3− and a resistance barrier to CO2 efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells. 相似文献
3.
Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942 总被引:5,自引:4,他引:1
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Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (Ci) conditions (1% CO2; pH 8) were found to be photosynthetically dependent on exogenous CO2. This was judged by the fact that they had a similar photosynthetic affinity for CO2 (K0.5[CO2] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for Ci measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO2 in high Ci cells. The addition of 200 micromolar EZ to high Ci cells increased K0.5(CO2) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low Ci cells (grown at 30 microliters CO2 per liter of air) were less sensitive to EZ. EZ inhibition in high and low Ci cells was largely relieved by increasing exogenous Ci up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO2 usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO2 usage. High Ci cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO2 usage in high Ci cells of Synechococcus PCC7942 may be due to inhibition of a `CA-like' function associated with the CO2 utilizing Ci pump or due to inhibition of an internal CA activity, thus affecting CO2 supply to ribulose bisphosphate carboxylase-oxygenase. 相似文献
4.
A high CO2-requiring mutant of Synechocystis PCC6803 (G3) capable of Ci transport but unable to utilize the intracellular Ci pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM
CO2-concentrating mechanism
- Ci
inorganic carbon
- HCR
high CO2-requiring
- kbp
kilobase pair
- ORF
open reading frame
- Rubisco
ribulose 1,5-bisphosphate carboxylase-oxygenase gene
- SSC
sodium chloride and sodium citrate
- WT
wild-type 相似文献
5.
Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this
protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially
either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem
II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor
side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from QA, gave a decreased O2 evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed
that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These
results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas 总被引:5,自引:5,他引:0
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Inorganic carbon (Ci) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% CO2-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (Ci) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO2-grown cells also accumulated some Ci, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high CO2-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO2-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase. 相似文献
7.
When cells of Anacystis nidulans strain R2 grown under high CO2 conditions (3%) were transferred to low CO2 conditions (0.05%), their ability to accumulate inorganic carbon (Ci) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO2 adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the Ci-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high Ci-accumulating capability. The incorporation of [35S]sulfate into membrane proteins was greatly reduced during low CO2 adaptation. Radioautograms of the 35S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active Ci transport by A. nidulans strain R2 and its synthesis under low CO2 conditions leads to high Ci-transporting activity. 相似文献
8.
9.
Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO2 总被引:2,自引:0,他引:2
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E. Marco I. Martinez M. Ronen-Tarazi M. I. Orus A. Kaplan 《Applied microbiology》1994,60(3):1018-1020
Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO2 for growth. 相似文献
10.
Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria 总被引:4,自引:4,他引:0
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Cells of a high CO2-requiring mutant (E1) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO2 exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO2 fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO2 uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO2 from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO2 uptake by the cells and the amount of CO2 released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO2-exchange profile. The COS concentration required for 50% inhibition of CO2 uptake was about 25 micromolar when the concentration of inorganic carbon (Ci) in the medium was 60 micromolar; higher Ci concentrations reduced the inhibitory effect of COS. Measurement of Ci uptake in E1 cells by a silicone oil centrifugation method also indicated marked reduction of the activities of 14CO2 and H14CO3− uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of Ci transport. 相似文献
11.
Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity 总被引:8,自引:6,他引:2
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In high inorganic carbon grown (1% CO2 [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO2 uptake and to reduce the final internal inorganic carbon (Ci) pool size reached. The relationship between CO2 fixation rate and internal Ci concentration in high Ci grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High Ci grown cells readily took up CO2 but had little or no capacity for HCO3− uptake. These cells appear to possess a CO2 utilizing Ci pump that has a CA-like function associated with the transport step such that HCO3− is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low Ci grown cells possess both CO2 uptake and HCO3− uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO2 and HCO3− uptake (such as the Ci pump) may have been affected. The inhibitor had no apparent effect on internal CO2/HCO3− equilibria (internal CA function) in low Ci grown cells. 相似文献
12.
Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome 总被引:11,自引:7,他引:4
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A total of 24 high CO2-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO2, on agar media, but are incapable of growth at air levels of CO2. All the mutants were able to accumulate inorganic carbon (Ci) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO2. On the basis of the rate of Ci release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast Ci release, normal internal Ci pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of 18O from 18O-labeled CO2, 1% CO2 requirement for growth in liquid media, normal affinity of carboxylase for CO2, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow Ci release, increased internal Ci pool, normal CA activity in crude extracts, normal internal 18O exchange, a 3% CO2 requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO2, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO3− to CO2 inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H+ import during photosynthesis. 相似文献
13.
Eiji Suzuki Hideya Fukuzawa Shigetoh Miyachi 《Molecular & general genetics : MGG》1991,226(3):401-408
Summary In a temperature-sensitive, high CO2-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis. 相似文献
14.
Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942 总被引:12,自引:5,他引:7
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Intact cells and crude homogenates of high (1% CO2) and low dissolved inorganic carbon (Ci) (30-50 microliters per liter of CO2) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of 18O from CO2 to H2O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low Ci cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO2. This activity is most apparent in the light, where 18O-labeled CO2 species are being taken up by the cells but the effluxing CO2 has lost most of its label to water. In the dark, low Ci cells catalyze the depletion of the 18O enrichment of CO2 and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the Ci pump and the Ci pump is proposed as a potential site for the exchange of 18O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low Ci cells. This was inhibited by ethoxyzolamide with an I50 of 50 to 100 micromolar in both low and high Ci cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg2+ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction. 相似文献
15.
The aim of this study was to express the zwf gene of Synechococcus sp. PCC 7942 in zwf mutant Escherichia coli DF214 cells and to analyse glucose-6-phosphate dehydrogenase (G6PDH) activity. Initially, mutant cells were transformed with plasmid pNUT1 containing a Synechococcus sp. PCC 7942 zwf gene with a 1 kb upstream region that is expected to contain promoter elements. Transformant DF214 cells were not complemented by this fragment in a glucose minimal medium, nor did they exhibit statistically meaningful G6PDH activity. Therefore, the zwf gene was cloned in the lac operon to express the Zwf as a fusion protein; this yielded the construct pSG162. The pSG162 transformant E. coli DF214 cells were complemented in a glucose minimal medium, indicating that cyanobacterial Zwf protein fused with the part of LacZ′ polypeptide, enabling the cells to utilize glucose via the oxidative pentose phosphate pathway. Compared with wild-type E. coli cells, approximately ten times more G6PDH activity was measured in transformant cells. This indicated that the Synechococcus sp. PCC 7942 zwf gene was expressed under the control of the E. coli lac promoter as a fusion protein and the zwf product was converted into an active G6PDH form. Analyses was also carried out to determine whether dithiothreitol (DTT) was an in vitro reducing agent affected the enzyme activity, as was previously reported for this cyanobacterial strain. The results showed no variation in enzyme activity in the reduced assay conditions. Therefore, the zwf mutant E. coli strain DF214 was found to provide a rapid system for analysis of cyanobacterial G6PDH enzymes, but not for the redox state analysis of this enzyme. 相似文献
16.
Jared M. Fraser Sarah E. Tulk Jennifer A. Jeans Douglas A. Campbell Thomas S. Bibby Amanda M. Cockshutt 《PloS one》2013,8(3)
Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b6f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes. 相似文献
17.
Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942 总被引:2,自引:2,他引:0
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Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO2 and HCO3− transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O2-evolution studies. The Ki for COS inhibition was 29 micromolar for CO2 transport and 110 micromolar for HCO3− transport. These results support a model of inorganic carbon transport with a central CO2 pump and an inducible HCO3− utilizing accessory protein which supplies CO2 to the primary pump. 相似文献
18.
19.
20.
Hideo Fukuda Miho Sakai Kazuhiro Nagahama Takao Fujii Masayoshi Matsuoka Yorinao Inoue Takahira Ogawa 《Biotechnology letters》1994,16(1):1-6
Summary Bioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringae pv.phaseolicola PK2 was cloned and expressed in the cyanobacteriumSynechococcus PCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivo ofSynechococcus PCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringae pv.phaseolicola PK2 was one-fifth of that ofE. coli JM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcus PCC7942 R2-SPc. 相似文献