Isolation and Characterization of High CO2-Requiring-Mutants of the ...

Received for publication February 21, 1989 and in revised form April 11, 1989

Plant Physiol. (1989) 91, 514-525 0032-0889/89/91/0514/1 2/$01 .00/0

Isolation and Characterization of High CO2-RequiringMutants of the Cyanobacterium Synechococcus PCC7942' Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO2 within the Carboxysome G. D. Price and M. R. Badger* Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, P. 0. Box 475, Canberra City, A.C. T. 2601, Australia (3, 12). In the past decade or so it has been possible to identify at least four key components of the CO2 concentrating mechanism in cyanobacteria (3). These are: (a) an active transport system for inorganic carbon (Ci) situated on the plasma membrane, (b) a means of coupling photosystem I energy to the C1 transporter, (c) a system for reducing the wasteful leakage of CO2 from the site of carboxylation, and (d) catalysis of the rate of CO2 production inside the cell through the use of the enzyme, CA. Recently it has been necessary to add a fifth component to this list; namely, a key role for the carboxysome. The carboxysome is a polyhedral body which is surrounded by a protein shell or coat and contains most, if not all, of the cells Rubisco (7). It now appears likely that the carboxysome acts as a microcompartment where CO2 supply to Rubisco can be raised through the action of localized CA activity (18). An essential feature of the carboxysome in this role would be the ability to provide a leak barrier to CO2 efflux from this compartment ( 19). The first suggestion of this type of role for the carboxysome came from experiments by Coleman et al. (8) who showed that carboxylase activity in intact carboxysomes was less sensitive to the oxygenase reaction than naked Rubisco enzyme. More recently, Reinhold et al. (19) have produced a theoretical quantitative model for photosynthesis and Ci fluxes in cyanobacteria that attributes the carboxysome with the property of having low permeability to gases like CO2 and 02 but having relatively high permeability to ions like HCO3-. This property would allow HCO3- to enter the carboxysome where CO2 would be generated through catalysis with CA, in a relatively gas tight environment. The best evidence that carboxysomes are involved in the CO2 concentrating mechanism comes from a recent experiment where active human CA enzyme was expressed in the cytosol of Synechococcus PCC7942 cells (18). This high activity CA caused a 'short-circuit' in Ci accumulation by bringing about a dramatic increase in the CO2 efflux rate. This experiment confirmed not only that HC03- is the species supplied to the cytosol by the Ci pump, but that the normal CO2 leak barrier in the cell must reside at the level of the carboxysome. The human CA experiment also indicates that cyanobacterial CA must be exclusively located in the carboxysome in order to minimize the rate of CO2 generation in the cytosol and

ABSTRACT A total of 24 high C02-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% C02, on agar media, but are incapable of growth at air levels of CO2. All the mutants were able to accumulate inorganic carbon (Cl) 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 C, 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 C, release, normal intemal C, pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced intemal exchange of 180 from 110-labeled C02, I % CO2 requirement for growth in liquid media, normal affinity of carboxylase for C02, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C, release, increased intemal C, pool, normal CA activity in crude extracts, normal intemal ISO exchange, a 3% CO2 requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for C02, 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 HC03- 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 11 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. Altematively the type 11 mutants may have a lesion in their capacity for H+ import during photosynthesis.

Cyanobacteria possess a very efficient 'CO2 concentrating mechanism' which functions to maximize the intracellular CO2 concentration around the CO2 fixing enzyme, Rubisco2 ' This work was supported by a National Research Followup (to G. D. P.) awarded by the Australian Government Department of Education, Employment and Training. 2 Abbreviations: Rubisco, ribulose bisphosphate carboxylase-oxygenase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; '4CDMO, 5,5-dimethyl[2-'4C]oxazolidine-2,4-dione; CA, carbonic anhydrase; Ci, dissolved inorganic carbon; EZ, ethoxyzolamide; Ko.5, concentration required for half-maximal response; WT, wild type.

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thus minimize CO2 leakage. It appears that either the protein shell surrounding the carboxysome or a centralized arrangement of CA inside the carboxysome is responsible for the CO2 leak barrier. In Synechococcus PCC7942 it is now known that CA is associated with the carboxysomal fraction but the exact location has yet to be confirmed (5). The characterization of cyanobacterial mutants defective in components of the CO2 concentrating mechanism presents a potentially powerful tool for understanding the molecular aspects of the process. So far, the isolation and characterization of only one type of mutant has been published by Marcus et al. (13) and Ogawa et al. (15). These two mutant isolates of Synechococcus PCC7942 accumulate Ci but appear to be unable to utilize intracellular HC03- for the generation of intracellular CO2. The mutant isolated by Ogawa et al. (15) has an added lesion which results in an inability to induce the 'low Cj' phenotype. The purpose of the present study was to undertake the isolation and characterization of a large number of high CO2requiring-mutants of Synechococcus PCC7942 affected in any one of the five basic components of the CO2 concentrating mechanism. We report the isolation and partial characterization of 24 mutants representing two extreme phenotypes that are able to accumulate Ci but would appear to be defective in their ability to generate CO2 within the carboxysome. MATERIALS AND METHODS Growth of Cyanobacteria

Synechococcus PCC7942 (Anacystis nidulans R2) was grown in BG 11 media (20) buffered to pH 8 with 1O mM BTP-HCI and gassed with air, 1% (v/v) CO2, 3% CO2, or 30 AL.L-' C02, as previously described (16). High C02-requiring-mutants were grown at 1 % or 3% CO2. Plate colonies were grown on 1% agar/BGl 1 media buffered with 50 mM Tes-NaOH and containing 5 mm sodium thiosulphate (TTES plates). These plates were sealed in large plastic boxes with transparent lids and gassed with humidified air or 1% CO2 in air by means of an inlet and outlet port. Plates were incubated at 30°C and a light intensity of 30 ,umol . m2 * s'. Mutagenesis and Selection of High C02-RequiringMutants The following protocol was adapted from one developed by T. Carlson and J. Pierce (personal communication). Synechococcus PCC7942 cells were grown in liquid media (1% C02) to a cell density of 108 cells ml-' (OD730 = 1.01.1), harvested by centrifugation and resuspended in sterile 30 mm phosphate buffer (pH 7.0) to a cell density of 2 x l09 cells- ml-'. For the mutagenesis step, 1 mL cells was combined with 1 mL phosphate buffer containing 0.4 M ethylmethyl sulphonate. The cells were then mixed and incubated, in the dark, for 45 min at 37°C. The mutagen was then inactivated by the addition of 10 mL of sterile, freshly prepared 5% sodium thiosulphate (pH 8). The cells were vortexed, collected by centrifugation (4000g), and resuspended in 1 mL of unbuffered BG 1. This step was repeated and cells were resuspended at 10-2 and 10-3 dilutions. (After mutagenesis, the

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10-' dilution had approximately 104 viable cells.ml-' on plates incubated at 1 % C02). A 100 L aliquot of each dilution was plated out in 3 ml top agarose (0.5% in Tes buffered BGl 1) onto a TTES plate containing 10 mL agar. When the top agarose had set a further 3 mL of agarose was added to provide a protective layer over the cells. Five plates of each dilution were set up. Plates were incubated under 1% CO2 conditions (low light; 10 ,Umol. m-2. s') for 18 h and then transferred to air for 3 d to deplete internal carbon reserves in putative high C02requiring-mutants. After this period, ampicillin (0.5 mL) was added to each plate to a final concentration of 40 gg ml-' and the plates were kept at air for a further 18 h (30 ,E*m-2 s-'). After this ampicillin enrichment step the remaining ampicillin was inactivated by the addition of 1.8 units of filter sterilized penicillinase (Sigma, USA) per plate and the plates were returned to air for 8 d. At this point, any WT colonies that had survived the enrichment step were marked and the plates were then transferred to 1% CO2 until putative high C02-requiring-mutants appeared (8-9 d). Putative mutant colonies were isolated as agar plugs with sterile Pasteur pipets and resuspended in 100 gL of BG 11 media. A total of 180 colonies were isolated from two 'l0-4' plates and these were rescreened on duplicate plates under air (nonpermissive) and 1% CO2 (permissive) conditions. Twenty-four isolates were confirmed as being 1% C02-requiring-mutants. Each mutant represented a separate mutagenic event since each colony had been kept spatially isolated since the time of mutagenesis.

180 Exchange Kinetics of Intact Cells 180 exchange kinetics were performed as described previously (5, 17). Cells were harvested by centrifugation (4000g for 10 min at 25°C) and resuspended in CO2 free growth medium buffered with 20 mm BTP-HCl (pH 8.0) to a final concentration of 20 to 50 ug Chl-ml-'. Two mL of the cell suspension was placed into the glass cuvette in the dark at 30°C together with 2 ,uL of 1.0 M NaH'3C'803 (1 mm final concentration). The cuvette was connected to a mass spectrometer (VG Micromass 6, Winsford, England) via a membrane inlet. Recording of the labeled masses (masses 49, 47, and 45) was commenced in the dark and monitored against time during dark -- light -- dark transients.

Cl Accumulation Time-Courses Time courses for internal Ci accumulation were performed as described previously (17) by use of a glass cuvette connected to a mass spectrometer, as described above. Cells suspended in BG 11 + 20 mm BTP (pH 8.0) were placed in the cuvette at 30°C and bovine carbonic anhydrase (Sigma, USA) was added to a final concentration of 0.1 mg. ml-'. NaHCO3 was added to a final concentration of 1 mm and the mass 44 signal monitored. A calibration for Ci concentration was achieved by injecting known amounts of NaHCO3 into the cuvette. Internal cell volume was assumed to be 60 LI -mg-' Chl (17). Cell Conductance Measurements Calculation of cell conductance to CO2 was carried out as previously described (6). This method is based on estimating

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the initial slope of Ci release in the dark immediately following an accumulation of C, in the light. This method requires measurement of the internal C, pool at the end of the light period and a calculation of the internal [CO2] assuming an internal pH of 8.0, a pKa of 6.12, and that C, species are in rapid equilibrium due to the presence of carbonic anhydrase inside the cell. The cell surface area was assumed to be 2500 cm2 * mg Chl'. The accumulation of C1 and the rate of release in the dark was estimated from time courses similar to those shown in the results section.

Carbonic Anhydrase Measurements in Crude Extracts Crude homogenates of 1% CO2 grown cells were prepared as previously described (5), except that the extraction buffer was 100 mM EPPS-NaOH (pH 8.0), 10 mM MgSO4, 1 mM EDTA, 0.5 mm PMSF, 10 ,M leupeptin (Boehringer Mannheim), 10 mm DTT, and 10 Ag mL-' DNAse. Extracts were prepared in 1 to 2 mL of extraction buffer at a Chl concentration of 400 to 700 ug.ml1'. Assays for CA activity were made at 30°C by measuring the rate of decline in the 180 enrichment of CO2 species in solution as previously described (5). The assay medium contained 100 mM Tricine-NaOH (pH 8.0), 10 mM MgSO4, and 1 mM NaH'3C'803. Crude extracts were injected into the cuvette to start the assay. Final Chl density in the cuvette was 50 to 150 jAg.mL-'. In this assay system one unit of carbonic anhydrase activity is equivalent to a 100% stimulation of the first order rate constant for the loss of label from doubly labeled CO2 species.

Oxygen Electrode Measurements

Photosynthetic response to external Ci were measured in an 02 electrode as previously described (16). Chl content was determined by the method of Wintermanns and de Mots (24). Silicone Oil Centrifugation/Filtration Experiments To test mutants for their ability to induce HC03- uptake (low Ci cell phenotype), CO2 uptake and HC03- uptake were measured in 1% CO2 grown cells before and after a 24 h period of growth at 30 ,iLu L' CO2. The uptake of 14CO2/ H'4CO3- was measured using the silicone oil filtration method, as previously described (4, 17). Cells were harvested by centrifugation and resuspended in BG 11 media buffered with 20 mm BTP-HCI (pH 8) to a Chl density of 2 to 4 ,g. ml-'. The buffer had been previously sparged with C02-free air to achieve a Ci concentration of 1

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Figure 9. Theoretical effect of increasing the conductance of the carboxysomal coat to C02 diffusion. Other parameters of the model were kept constant (see "Materials and Methods") while the C02 conductance of the coat was varied from 1 0-5 to 10-1 cm * s51. The responses of both photosynthesis (A) and cytosolic Ci (B) to extemal

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efflux of singly '80-labeled CO2 (mass 47) (Fig. 5). During Ci efflux in the dark, however, there is evidence of some efflux of mass 47 which points to there being slower exchange inside the cells (Fig. 5). Thus, the exact nature of C1 exchange processes in the cell is ambiguous but, clearly, CO2 efflux in the dark is much slower than in WT cells. It is possible that the leakage of CO2 out of the cell is so slow that the C1 pump can recycle most of the leaked CO2. In this case the C1 pump, which binds CO2 and releases it internally as HCO3- (16, 17, 23), would itself be responsible for exchanging 180 out of CO2. However, this would not appear to be a reasonable explanation since there is clearly efflux of unlabeled CO2 in the light (Fig. 5). Another feature of type II mutants is that they have high levels of carboxylase activity (Table II) and this is especially so when cells are grown to high Chl density under suboptimal CO2 conditions (i.e. 1% CO2) (Table III). These mutants, have correspondingly increased numbers of carboxysomes, especially when grown at 1% CO2 (Fig. 6). It is known that the 'low Ci' state in WT cells is accompanied by an increase in carboxysome numbers (22). Thus, it is likely that increased carboxylase activity and carboxysome numbers are a pleiotropic effect related to low CO2 levels within the carboxysome because the increase in carboxylase activity is exacerbated when cells are grown at 1% C02 rather than 3% CO2 (Fig. 6). The type of scenario that comes closest to explaining the type II phenotype is one where carboxysomal CA is postulated to be totally absent from the cell. Modeling this situation in Figure 10 predicts that the cells would: (a) accumulate higher than normal levels of Ci, (b) have a slow leak rate, and (c) require extremely high levels of CO2 to overcome the low conductance barrier of the carboxysome shell in order to saturate photosynthesis. However, this explanation does not fit well because normal levels of CA activity can be assayed in extracts from type II mutants (Table II) and there is evidence from 180 exchange studies that indicates there may be rapid interchange of C1 species inside the cell (Fig. 5). An alternative proposal for the type II mutants is a situation where a lesion in active proton import (Na+/H+ antiport) would limit photosynthesis by slowing the rate of CO2 generation within the carboxysome. This situation should be equivalent to a scenario postulating the total removal of CA from the cell (see above). This suggestion would depend on whether the stoichiometric H+ efflux during Ci accumulation (14) is tightly coupled to CO2 uptake and whether it remains that way during steady state photosynthesis. If H+ efflux and CO2 uptake are tightly coupled, then there would be a need to import H+ at the same rate as CO2 fixation. Intuitively, a lesion in the capacity for H+ import might cause cytoplasmic pH (pHj) to become excessively high, but this might not be obligatory. Unfortunately, it is not yet possible to model pH, changes associated with the CO2 concentrating mechanism in cyanobacteria. Measurements of pH, in type II mutants (and type I) indicates that pH, values under standard conditions (light on; 1 mm Ci), although slightly high, are not significantly higher than in WT cells (Table V). Clearly there are still problems in trying to understand the type II phenotype and work in this direction is still continuing. The type II mutant phenotype appears to be a new mutant

Plant Physiol. Vol. 91, 1989

class that is quite distinct from the E, and RK1 mutants (13, 15).

CONCLUSION We have partially characterized two types of high C02requiring-mutants of the cyanobacterium, Synechococcus PCC7942. Both phenotypes accumulate Ci to levels at or above that of WT cells but appear to be incapable of generating CO2 within the carboxysome. Further studies are still required to pinpoint the molecular basis for each mutant type reported here. It is interesting that out of 24 isolates no mutants defective in the ability to accumulate Ci were isolated. This may indicate one of the following: (a) that a 'pumpless' mutant is lethal, (b) that our current mutant screening approach (1% C02) is inappropriate, or (c) that the gene(s) for the Ci pump may be members of multigene groups. ACKNOWLEDGMENTS We are indebted to Dr. John Andrews for commenting on the manuscript, Mr. Ian Duff (ANU TEM unit) for sectioning and photography of EM samples, and Ms. Susan Kirby for expert technical assistance. Thanks are also due to the staff of the ANU TEM unit for general technical support. LITERATURE CITED 1. Andrews TJ, Abel KM (1981) Kinetics and subunit interactions of ribulose bisphosphate carboxylase-oxygenase from the cyanobacterium Synechococcus sp. J Biol Chem 256: 8445-8451 2. Badger MR (1980) Kinetic properties of ribulose 1,5-bisphos-

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phate carboxylase/oxygenase from Anabaena variablis. Arch Biochem Biophys 201: 247-254 Badger MR (1987) The CO2 concentrating mechanism in aquatic phototrophs. In MD Hatch, NK Boardman, eds, The Biochemistry of Plants: A Comprehensive Treatise, Vol 10, Photosynthesis. Academic Press, New York, pp 219-274 Badger MR, Gallagher A (1987) Adaptation of photosynthetic CO2 and HCO3- accumulation by the cyanobacterium Synechococcus PCC6301 to growth at different inorganic carbon concentrations. Aust J Plant Physiol 14: 189-210 Badger MR, Price GD (1989) Carbonic anhydrase activity associated with the cyanobacterium Synechococcus PCC7942. Plant Physiol 89: 51-60 Badger MR, Bassett M, Comins HN (1985) A model for HC03accumulation and photosynthesis in the cyanobacterium Synechococcus sp. Plant Physiol 77: 465-471 Codd GA, Marsden WJN (1984) The carboxysomes (polyhedral bodies) of autotrophic prokaryotes. Biol Rev 59: 389-422 Coleman JR, Seemann JR, Berry JA (1982) RuBP carboxylase in carboxysomes of blue-green algae. Carnegie Inst Wash Year Book 81: 83-87 Dzelzkalns VA, Owens GC, Bogorad L (1984) Chloroplast promoter driven expression of the chloramphenicol acetyl transferase gene in a cyanobacterium. Nucleic Acids Res 12: 89178925 Gantt E, Conti SF (1969) Ultrastructure of blue-green algae. J Bacteriol 97: 1486-1493 Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol 153: 215-231 Kaplan A, Badger MR, Berry JA (1980) Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variablis: response to external CO2 concentration. Planta 149: 219-226 Marcus Y, Schartz R, Friedberg D, Kaplan A (1986) High CO2 requiring mutant of Anacystis nidulans R2. Plant Physiol 82: 610-612

HIGH C02-REQUIRING-MUTANTS OF SYNECHOCOCCUS PCC7942 14. Ogawa T, Kaplan A (1987) The stoichiometry between CO2 and H+ fluxes involved in the transport of inorganic carbon in cyanobacteria. Plant Physiol 83: 888-891 15. Ogawa T, Kaneda T, Omata T (1987) A mutant of Synechococcus PCC7942 incapable of adapting to low CO2 concentration. Plant Physiol 84: 711-715 16. Price GD, Badger MR (1989) Ethoxyzolamide inhibition of C02dependent photosynthesis in the cyanobacterium Synechococcus PCC7942. Plant Physiol 89: 44-50 17. Price GD, Badger MR (1989) Ethoxyzolamide inhibition of C02 uptake in the cyanobacterium Synechococcus PCC7942 without apparent inhibition of internal carbonic anhydrase activity. Plant Physiol 89: 37-43 18. Price GD, Badger MR (1989) Expression of human carbonic anhydrase in the cyanobacterium Synechococcus PCC7942 creates a high CO2-requiring-phenotype: evidence for a central role for carboxysomes in the CO2 concentrating mechanism. Plant Physiol 91: 505-513 19. Reinhold L, Zviman M, Kaplan A (1987) Inorganic carbon fluxes

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and photosynthesis in cyanobacteria-a quantitative model. In J Biggens, ed, Progress in Photosynthesis, Vol IV. Martinus

Nijhoff, Dordrecht, Netherlands, pp 6.289-6.296 20. Ripka R, Waterbury JB, Stanier RY (1981) Isolation and purification of cyanobacteria; Some general principles. In MP Starr, H Stolp, HG Truper, A Balows, HG Schlegel eds, The Prokaryotes. Springer-Verlag, Berlin, pp 212-220 21. Smith FA, MacRobbie EAC (1981) Comparison of cytoplasmic pH and Cl- influx in cells of Chara corallina following C1starvation. J Exp Bot 32: 827-835 22. Turpin DH, Millar AG, Canvin DT (1984) Carboxysome content of Synechococcus leopoliensis (Cyanophyta) in response to inorganic carbon. J Phycol 20: 249-253 23. Volokita M, Zenvirth D, Kaplan A, Reinhold L (1984) Nature of the inorganic carbon species activity taken up by the cyanobacterium Anabaena variablis. Plant Physiol 76: 599-602 24. Wintermans JFGM, de Mots A (1965) Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim Biophys Acta 109: 448-453