Vol. 132, No. 3 Printed in U.S. A.
JOtJRNAL OF BACTERIOLOGY, Dec. 1977, p. 771-778 Copyright © 1977 American Society for Microbiology
Phycocyanin Synthesis and Degradation in the Blue-Green Bacterium Anacystis nidulans REGINALD H. LAU, MARGARET M. MAcKENZIE, AND W. FORD DOOLITTLE* Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7 Received for publication 16 August 1977
Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits; (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation. Blue-green bacteria owe their characteristic lans, a unicellular blue-green bacterium that colors to the biliproteins phycocyanin (PC) and contains only PC as the major biliprotein. Allen (in some species) phycoerythrin. These chro- and Smith (2) earlier characterized spectrophomophore-containing, accessory pigments cap- tometric changes under these conditions and ture much of the light energy essential for pho- concluded that nitrogen source (nitrate) starvatoautotrophic growth (4). At the same time, they tion results in loss of PC color (chlorosis), whereas restoration of nitrate promotes (after a can constitute up to 60% of the total cellular protein (4), and their syntheses must require a lag) rapid repigmentation, which must be comsignificant expenditure of that energy. It is pleted before cell growth can resume. Here, we clearly of selective advantage for blue-green bac- have asked (i) whether chlorosis indeed involves teria to control the rates of synthesis of PC and loss of PC apoprotein as well as loss of chromophycoerythrin apoproteins (and their cognate phore; (ii) whether de novo apoprotein synthesis chromophores) in ways that optimize, for any continues at a high relative rate during starvaenvironmental condition, functional phycobili- tion or is selectively depressed; (iii) whether the observed lag in repigmentation after nitrate resprotein content. Phycobiliprotein content, as measured spec- toration reflects a preferential lag in resumption trophotometrically, is indeed affected by (i) light of PC apoprotein synthesis; (iv) whether illumiquality (chromatic adaptation [3, 4, 14]), (ii) nation affects relative rates of PC apoprotein light quantity (9), and (iii) the availability of synthesis during repigmentation; and (v) whether C02, phosphate, and nitrate (2, 7, 8). However, the two PC apoprotein subunits (a, 16,000; /, spectrophotometric determinations reflect only 19,000 daltons [4]) are coordinately synthesized intact (chromophore-containing) PC content (or degraded) under these conditions. and do not differentiate changes effected at the MATERIALS AND METHODS level of apoprotein synthesis from those effected at the levels of protein turnover or chromophore Culture and labeling conditions. Wild-type A. attachment. Since it is commonly held (5, 6) nidulans was maintained as described previously (11, that blue-green bacteria are incapable of re- 12). One-liter cultures in the nitrate-containing mesponding to environmental variation by specific dium of Allen (1) were grown (except where noted) alterations in patterns of gene expression (pro- at 37°C under 5% C02-95% air in mechanically stirred Fernbach flasks that were 30 cm from four tein synthesis), it is especially important to char- 2.8-litercool-white fluorescent tubes. Cultures were laacterize, at the level of apoprotein formation, 40-W either with L-[4,5-3H (N)]leucine (New England beled molecular events during such pigmentation Nuclear Corp., Boston, Mass.) at 1 MCi and 0.01 I,g/ml changes. for two to four generations (10 to 20 h) or with L-[ UWe have chosen to examine alterations in 14C]leucine (New England Nuclear Corp) at 0.4 MCi intact-PC content, apoprotein content, and apo- and 0.3 Ag/ml for 20 min (pulse-labeling). Pigment and protein determination. For specprotein synthesis provoked by nitrogen source starvation and restoration in Anacystis nidu- trophometric determinations of PC, chlorophyll, and 771
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carotenoids, cells were harvested by centrifugation, frozen, and stored (I to 4 days) at -20'C. Chlorophyll and carotenoids were extracted from thawed cells with 2 to 5 ml of 80% acetone and measured at 665 and 460 nm, respectively (2). PC was determined in aqueous suspensions of acetone-extracted intact cells as described by Allen and Smith (2). All spectrophotometric values are expressed as the percentages of values obtained with samples taken at zero time (see Results). Protein content was determined as described by Ihlenfeldt and Gibson (8). Determination of 3H and '4C radioactivity in PC apoprotein: Method A. Samples were harvested by centrifugation, frozen for storage (1 to 4 days at -20'C), thawed, and suspended in :3.5 ml of lvsis buffer (11). Lysates obtained by passage through an Aminco French pressure cell at 1.1 x 10' kg/mi were freed from membranous debris bv cenitrifugation at 17,000 x g for 30 min (conditions that do not sediment biliproteins). Proteins were precipitated (30 min at room temperature) by the addition of trichloroacetic acid to 10%S, collected by centrifugation, washed twice with ethaniol, dried unlder vacuum, suspeended in sodium dodecyl sulfate (SDS)-containing sample buffer, heated to 100°C for 3 min, and loaded onto SDS-10% polyacrylamide gels, all as described previously (11). Molecular-weight-marker gels, loaded with phosphorylase a, bovine serum albumin, ovalbumin, pepsin, carbonic anhydrase, beta-lactoglobulin, cytochrome c, and/or purified A. nidulans PC (a generous gift of M. V. Laycock) were run simultaneously. For determinations of radioactivity, 1-mm gel slices were covered with 6 ml of toluene containing 57c P'rotosol (New England Nuclear Corp), 0.8K Omnifluor (New England Nuclear Corp.), and 0.3` water, shaken for 1 to 2 days at 37°C in polyethylene scintillation vials, and counted in a liquid scintillation counter programmed to correct for 4C crossover and quenchinig. Radioactivity in PC apoprotein a and /3 subunits and in total protein was quantitated by measuring areas under peaks on gel profiles (e.g., Fig. 5). Method B. For preparation of antisera, four rabbits were injected subcutaneously with approximately 1 mg of PC (showing only a and /3 subunit bands on SDS-polyacrylamide gels) in complete Freund adjuvant. Subsequent injections at 1, 2, 3, and 9 weeks were made in incomplete adjuvant, and rabbits were sacrificed bv heart puncture at 10 weeks. To 0.25-ml portions of cell lvsates prepared and free(d from membranous debris as described above, 24 ,ug of nonradioactive, purified PC (and, for experiments measuring PC apoprotein disappearance only, 12 ,l of a similarly prepared "standard lysate" of ["4C]leucine-labeled logarithmically grown cells) was added. Antiserum (0.25 ml) was then added, and the mixture was incubated for 20 min at 30°C and for 16 to 20 h at 4°C. Immunoprecipitates were collected by centrifugation at 27,000 x g for 5 min and washed twice with 5 ml of 0.05 M potassium phosphate (pH 7.0). Control experiments revealed that at least 90%/ of the added (nonradioactive) PC was precipitated under these conditions. Trichloroacetic acid precipitation, solubilization, gel electrophoresis, and determination of radioactivity in individual gel slices were performed as described above.
In experiments measuring disappearance of PC apoprotein (labeled with [rHlleucine before nitrate starvation), the ratio of 'H- to '4C-labeled, antibody-precipitated material migrating as PC was divided by the ratio of total "H- to 4C-labeled protein in the samples before immunoprecipitation (determined from profiles of S1)S-polyacrylamide gels of total soluble proteins). Since [ "'C]PC was a constant fraction of' the total "Clabeled (standard lysate) protein in all such mixtures, this procedure correcte(d for sample-to-sample differences in the efficiency of' cell lvsis, immunoprecipitate recovery, and gel loadiing. In experiments measuring the rate of PC apoprotein svnthesis after nitrate restoration (see Fig. 5 to , cells were labeled with [tH]leucine. starved, and then (at intervals after restoration of nitrate) labeled (2(1 min) with [14C]leucine. Relative incorporation of ['4Clleucine into PC apoprotein (corrected for variations in [r4Clleucine uptake, immunoprecipitation, and gel loading) was determined by dividing the ratio of 4C counts per minute to ~'H counts per minute in antibody-precipitated material migrating on gels as PC by the ratio 4C/ 'H in gels of total soluble protein. These relative values were multiplied by that fraction of total q'H counts per minute comprising PC in each sample (determined by method A). This last procedure yielded values for incorporation of '4C into PC apoprotein (as percent total 14C incorporation) and corrected for possible variations in relative [3H1PC content resulting from continued in vivo degradation of PC or turnover of other (non-PC) IH-labeled protein after nitrate restoration. Method B also permitted accuracy in determining 14C incorporation into PC in samples labeled early after nitrate restoration, when this protein comprised too small a fraction of total labeled material for determination by method A.
RE,SULTS Loss of PC apoprotein during nitrate starvation. Figure 1 illustrates results of a typical nitrate starvation-restoration experiment. Cells growing exponentially in nitrate-containing, [ XH]leucine-supplemented medium were washed and suspended at zero time in nitratefree (or, as a control, nitrate-containing) mediumn and incubated under growth conditions. Nitrate was restored to the starved culture at 63.5 h. At intervals during and after starvation, cells were harvested by centrifugation and frozen for subsequent spectrophotometric determination of chlorophyll, carotenoids, and intact (chromophore-containing) PC, and for independent determination of relative 3H-labeled PC apoprotein content. Turbidity at 750 nm was monitored in control and starved cultures throughout the experiment. Loss of spectrophotometrically measurable PC began soon after resuspension in nitrate-free medium, while turbidity, carotenoid, and chlorophyll levels continued to increase for at least 35 h (Fig. 1). (Continued increases in all four values were observed in the nitrate-containing
PHYCOCYANIN SYNTHESIS AND DEGRADATION
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FIG. 1. Spectrophotometric changes during nitrate starvation and after nitrate restoration. A logarithmically growing culture was washed and suspended (at zero time) in nitrate-free medium. Contents (per milliliter) of (0) PC, (A) chlorophyll, and (A) carotenoids, and turbidity (0) at 750 nm were followed. Nitrate was restored at 63.5 h (arrow). Inset shows results with a control (nitrate-containing) culture incubated in parallel. All values are expressed as percentages of values obtained at zero time.
control culture [inset] until this culture entered stationary phase at approximately 60 h.) These results are in qualitative agreement with those of Allen and Smith (2), although loss of PC absorbance was less rapid and complete than in their experiments. Loss of PC absorbance was paralleled by loss of apoprotein, as determined in two ways. In the first, method A, loss of 3H from PC (labeled before starvation) was quantitated by dividing 3H radioactivity in bands corresponding to PC apoprotein a and ,8 subunits by total radioactivity in all bands on 10% polyacrylamide gels of total soluble cellular proteins (for examples of such gels, see Fig. 5). Although other polypeptides must, of course, migrate with these subunits, the method can pretend to some accuracy, since (under present conditions) these two species alone comprise 10 to 20% of the total soluble protein. Values determined in this way are plotted (as a percentage of the values obtained immediately after nitrate removal) in Fig. 2.
773
In the second method (B), constant amounts of ['4C]leucine-labeled A. nidulans total soluble protein (prepared separately) and purified, carrier nonradioactive PC were added to portions of the same 3H-labeled (starved-cell) total protein preparations and precipitated with antiserum prepared in rabbits against purified A. nidulans PC. Immunoprecipitates were washed, solubilized, and resolved on 10% polyacrylamide gels. Gel profiles for samples taken at 0 and 63.5 h after starvation initiation are illustrated in Fig. 3. Values for immunoprecipitated 3H-labeled material migrating as PC on such gels were normalized (Materials and Methods) and are plotted (as percentages of values obtained immediately after nitrate removal) in Fig. 2. Rates of PC apoprotein disappearance derived by these two independent methods are in reasonable agreement with each other and with the rate of loss of spectrophotometrically measurable (chromophore-containing) PC. We conclude that chlorosis indeed involves apoprotein degradation as well as chromophore loss, although it is not possible to say (because of scatter in the data) how precisely these events are coordinated. It is possible to compare individual rates of loss of the two subunits of PC apoprotein, since these were at least partially resolved on SDS-polyacrylamide gels of immunoprecipitated material. Values of 3H counts per minute/14C counts per minute for , (peak slice) divided by 3H counts per minute/14C counts per
HOURS STARVATION
FIG. 2. Correlation of loss of spectrophotometrically measurable PC with loss of PC apoprotein subunits. Relative PC apoprotein content determined by SDS-gel electrophoresis of total soluble proteins (0, method A) or of proteins precipitated by antiserum directed against purified PC (0, method B). (O) Spectrophotometrically measurable PC (from Fig. 1). Values are expressed as percentages of values obtained at zero time. Inset: Ratio of PC a and ft subunits during starvation.
774
LAU, MACKENZIE, AND DOOLITTLE
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minute for a (peak slice) are shown in Fig. 2 and indicate that degradation of the two subunits is coordinate. De novo synthesis of PC during nitrate starvation. Rates of de novo synthesis of PC apoprotein during nitrate starvation were determined in an experiment (similar to that illustrated in Fig. 1) in which [3H]leucine-labeled, washed, and nitrate-starved cultures were pulselabeled with ['4C]leucine at 0, 3, 13, and 37 h after starvation initiation. After pulse-labeling (20 min), cells were harvested and lysed. Relative rates of PC apoprotein synthesis were taken as fractions of total 14C radioactivity migrating as a and subunits on polyacrylamide gels of total soluble protein. Although substantial incorporation of 14C into total protein was observed during nitrate starvation, in no sample (including that pulse-labeled immediately after starvation) did material migrating as PC comprise more than 1% of the total '4C-labeled material. (By contrast, up to 8% of the "4C-labeled protein from cultures pulse-labeled 10 h after nitrate restoration migrated as PC [see Fig. 7]). SDS-gel electrophoresis of immunoprecipitated samples also indicated that no higher-molecularweight (potential precursor) polypeptides with PC antigenic determinants were labeled during nitrate starvation. Thus, starvation either results in rapid preferential degradation of newly
synthesized PC apoprotein subunits or (as we feel more likely) specifically depresses expression of PC apoprotein structural genes. Resynthesis of PC after restoration of nitrate. Experiments such as that illustrated in Fig. 1 (and the earlier data of Allen and Smith [2]) suggest that, although PC levels (measured spectrophotometrically) begin to increase soon after readdition of nitrate, maximal rates of pigment accumulation are not achieved for several hours. A similar delay in the resumption of high rates of accumulation of spectrophotometrically measurable PC is seen in Fig. 4, which also illustrates the effects of (i) exclusion of light, addition of 3-(3,4-dichlorophenyl)-1,1-di(ii) methylurea (an inhibitor of photosystem II), and (iii) addition of chloramphenicol, all immediately before nitrate restoration. This delay could result from a lag in resumption of chromophore synthesis (with consequent accumulation of free apoprotein), a lag in resumption of protein synthesis affecting all cellular components, or a specific lag in the resumption of PC apoprotein synthesis. Similarly, the failure of unilluminated (or 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated) cells to accumulate spectrophotometrically measurable PC rapidly, even long after nitrate restoration, could reflect the overall depression of protein synthesis previously observed in darkened cells (11) or a specific effect
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proteins (see Materials and Methods). In samples from the illuminated culture pulselabeled soon after nitrate restoration, PC a and ,B subunits comprised a small fraction of total "C-labeled proteins and polypeptides of molec-7 ular weights between 20,000 and 80,000 (some, 0 but not all, of which comigrated with 3H-labeled E species) predominated (Fig. 5). ImmunoprecipiCL4 tated [14C]PC was similarly low (Fig. 6), and no higher-molecular-weight proteins (potential precursors) carrying PC antigenic determinants were detectable on gels of immunoprecipitates. 5 10 Percentages of "C-pulse-labeled material from illuminated and darkened cells precipitated HOURS AFTER NITRATE RESITRAIIUN FIG. 4. Resumption of accumulation of spectro- by anti-PC and migrating as PC on SDS-polyphotometrically measurable PC after restoration of acrylamide gels (method B, normalized as denitrate to a starved culture. A logarithmically grow- scribed in Materials and Methods) are plotted ing culture was washed, suspended in nitrate-free against time after nitrate restoration in Fig. 7. medium, and incubated until spectrophotometrically Values determined by method A are also shown measurable PC fell to about one-half of its original (for the illuminated culture only). Agreement value. At zero time, the culture was divided into four between values determined by these two indesubcultures, nitrate was restored to each, and PC pendent methods is good, although quantitation content was determined spectrophotometrically at intervals thereafter. Symbols: 0, control (illuminated) of areas under the peaks is obviously uncertain subculture; 0, unilluminated subculture; V, subcul- when relative PC apoprotein synthesis is low (e.g., panels A and B of Fig. 5), and we consider ture containing 100 ug of chloramphenicol per ml; A, subculture containing 10 4ug of 3-(3,4-dichloro- method B to be the more accurate. Figure 7 phenyl)-1,1-dimethylurea per ml. All values are ex- also presents, for both cultures, results of specpressed as percentages of values at the time of nitrate trophotometric measurements of intact (chrorestoration. mophore-containing) PC per milliliter of culture and determinations of total protein per milliliter on relative rates of chromophore and/or apopro(both presented as the percentages of values tein formation. To distinguish between these possibilities, ex- before nitrate removal). It should be realized periments like those illustrated in Fig. 5 to 7 that these latter are measures of total pigment were performed. [3H]leucine-labeled cells were and protein accumulation, whereas measurewashed and starved for nitrate, as before. After ments of percent "C incorporation into PC respectrophotometrically measurable PC levels flect relative rates of apoprotein synthesis. In illuminated cells, relative rates of PC apofell to about one-half their original value, nitrate protein synthesis rose 6- to 10-fold during the was restored (zero time), and the culture was divided into two subcultures, one maintained first 9 h after nitrate restoration, becoming conunder continuous illumination and the other stant (at about 8% of the total-soluble-protein maintained in darkness. At intervals, samples synthesis) shortly after the resumption of accumulation of spectrophotometrically measurable were removed and incubated (under the same illumination conditions) with ['4C]leucine for 20 PC and total protein. Comparisons of 14C/3H min before harvesting for lysis and protein ex- ratios in resolved a- and,8-subunit peaks on gels traction. Incorporation of ['4C]leucine into PC of immunoprecipitates revealed that the synthesis rates of the two PC apoprotein subunits are was determined as a fraction of the total incorporation by the two methods described above. coordinately controlled under these condiRepresentative profiles of 10% polyacrylamide tions. We cannot readily explain the failure of gels of total soluble proteins from samples taken relative PC apoprotein synthesis to reach levels at 1, 3, 9.5, and 15.5 h after nitrate addition comparable to those observed for PC apoprotein (illuminated culture) are shown in Fig. 5. Profiles content (ca. 20%, by method A) in steady-stateof radioactivity in gels of proteins precipitated labeled exponentially growing cultures. It may from these same samples with anti-PC are be relevant that the illuminated culture was shown in Fig. 6. For these latter, 3H radioactivity entering the stationary phase towards the end of the experiment illustrated in Fig. 5 to 7 and was normalized to constant recovery (relative to the 1-h sample) of [3H]PC a and ,f subunits; that, in other experiments (e.g., illustrated in '4C values were multiplied by the same factor Fig. 1), PC apoprotein synthesis rose from 2 to and corrected for differences in total 14C labeling 12% of the total protein synthesis during the by using values for total "4C and 3H in soluble first 10 h after nitrate restoration. Similarly, we :n
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before nitrate starvation and pulse-labeled with l4 C]leucine after nitrate restoration. An exponentially growing culture was labeled with ['Hjleucine for several generations, washed, and suspended in nonradioactive, nitrate-free medium. Nitrate was restored after spectrophotometrically measurable PC had fallen to about one-half of its original level, and incubation (under illumination) was continued for 17.5 h. At intervals. samples were withdrawn and pulse-labeled (20 min under illuminati'on) with [l4C/leucine. Cells were harvested, and the distribution of 'H and 14C radi'oactivities in total soluble proteins was analyzed as described in the text. Results are shown for samples pulse- labeled at I (A), 3 (B), 9.5 (C), and 15.5 (D) h after nitrate restorati'on. Numbers in panel (A) indicate molecular weights (xIO') as determined by compari'son with simultaneously run gels containing "marker" proteins of known molecular weights. Positions of phycocyanin apoprotetin a and fl subunits are i'ndicated. Symbols: O, 'H counts per minute; *, 14C counts per minute.
776
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VOL. 132, 1977
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FIG. 7. Relative rates of synthesis of PC apoprotein and absolute levels of spectrophotometrically measurable PC and total protein after nitrate restoration. Relative rates of PC apoprotein synthesis (i.e., percentage of total 14C-pulse-labeled soluble protein comprising PC a and ,B subunits) were determined by methods A (0) and B (0), as described in the text, and are plotted against time of initiation of pulse-labeling. Spectrophotometrically measurable PC (O) and protein (V) content (per milliliter of culture) were determined with portions of the same samples and are expressed as percentages of values determined immediately after the initial removal of nitrate. (A) Culture continuously illuminated; (B) culture darkened upon restoration of nitrate.
cannot exclude the possibility that PC apoprotein is considerably more stable than the bulk of polypeptides synthesized soon after nitrate restoration. This possibility cannot be tested
directly, since pulse-labels can only slowly be "chased" from internal pools by the addition of excess nonradioactive amino acid. However, 3H label (which was adequately chased in these experiments) was redistributed (resulting in a 50% increase in relative [3H]PC content) after nitrate restoration, thus indicating that some (non-PC) proteins are degraded during the recovery period. In unilluminated cells, PC apoprotein synthesis after nitrate restoration follows a complex course (Fig. 6). Early after restoration this protein constituted, in darkened cells, a larger fraction of total pulse-labeled material than in ffiluminated cells. The failure of darkened cells to accumulate spectrophotometrically measurable PC thus cannot be attributed to lowered expression of PC apoprotein structural genes and can be adequately explained by the overall depression in protein synthesis reported previously (11) and apparent in Fig. 7. DISCUSSION We have confirmed a proposal (often an implicit assumption) made in earlier work dealing with pigmentation changes in blue-green bacteria: that spectrophotometric determinations of PC reflect (at least in the case of nitrate starvation and restoration) events at the level of apoprotein degradation and synthesis. (Bennett and Bogorad have provided similar evidence for
778
LAU, MACKENZIE, AND DOOLITTLE
changes in relative rates of PC and phycoerythrin apoprotein synthesis during chromatic adaption in Fremyella diplosiphon [3].) It seemed to us particularly important to provide such confirmation for nitrate starvation-induced chlorosis, since blue-green bacteria do possess other nitrogen stores (cyanophycin granules [10]) and the wholesale dismantling of the lightharvesting apparatus may seem an inappropriate initial response to nitrogen limitation. We can now conclude that (i) nitrate starvation-induced chlorosis is accompanied by coordinate loss of the two PC apoprotein subunits; (ii) during starvation, de novo PC apoprotein synthesis is specifically depressed; (iii) this specific depression persists for several hours after nitrate is restored (a- and /3-subunit syntheses recover in a coordinate fashion and reach maximal rates at about 10 h, shortly after the resumption of accumulation of total protein and spectrophotometrically measurable PC); and (iv) both rates of overall protein formation and relative rates of PC apoprotein synthesis are affected by light. These results should be viewed in the context of the hypothesis, put forward by Carr and his collaborators (5, 6), that blue-green bacteria are deficient in controls exerted at the level of gene expression and that the relative inability of these organisms to use potentially metabolizable exogenous sources of carbon and energy other than CO2 and light stems from this deficiency. It indeed appears that exogenous metabolites which are not normally required for (and which do not normally greatly stimulate) photoautotrophic growth do not evoke changes in specific activities of enzymes involved in their metabolism (5, 6, 12). However, environmental stimuli to which the growth of these organisms does respond (light, C02, phosphate, and nitrate) markedly influence patterns of protein synthesis (8, 11, 13), and it is simplest to attribute these specific environmental effects to controls operating at the level of gene expression.
J. BACTERIOL. ACKNOWLEDGMENTS This work was supported by grant MT 4467 from the Medical Research Council of Canada. We are very grateful to M. V. Laycock for samples of purified phycocyanin and to R. A. Singer for helpful discussions.
LITERATURE CITED 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 2. Allen, M. M., and A. J. Smith. 1969. Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69:114-120. 3. Bennett, A., and L. Bogorad. 1973. Complementary chromatic adaptation in a filamentous blue-green alga. J. Cell Biol. 58:419-435. 4. Bogorad, L. 1975. Phycobiliprotein and complementary chromatic adaptation. Annu. Rev. Plant Physiol. 26: 369-401. 5. Carr, N. G. 1973. Metabolic control and autotrophic physiology, p. 39-65. In N. G. Carr and B. A. Whitton (ed.), The biology of the blue-green algae. Blackwell Scientific Publications, Oxford. 6. Delaney, S. F., A. Dickson, and N. G. Carr. 1973. The control of homoserine-O-transsuccinylase in a methionine requiring mutant of the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 79:89-94. 7. Eley, J. H. 1971. Effect of carbon dioxide concentration in the blue-green alga Anacystis nidulans. Plant Cell Physiol. 12:311-316. 8. Ihlenfeldt, M. J. A., and J. Gibson. 1975. Phosphate utilization and alkaline phosphatase activity in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102: 22-28. 9. Jones, L. W., and J. Myers. 1965. Pigment variations in Anacystis nidulans induced by light of selected wavelengths. J. Phycol. 1:7-14. 10. Simon, R. D. 1973. Measurement of cyanophycin granule polypeptide content in the blue-green alga Anabaena cylindrica. J. Bacteriol. 114:1213-1216. 11. Singer, R. A., and W. F. Doolittle. 1975. Control of gene expression in blue-green algae. Nature (London) 253:650-651. 12. Singer, R. A., and W. F. Doolittle. 1975. Leucine biosynthesis in the blue-green bacterium Anacystits nidulans. J. Bacteriol. 124:810-814. 13. Stevens, S. E., Jr., and C. Van Baalen. 1974. Control of nitrate reductase in a blue-green alga: the effects of inhibitors, blue-light, and ammonia. Arch. Biochem. Biophys. 161:146-152. 14. Tandeau de Marsac, N. 1976. Occurrence and nature of. chromatic adaptation in cyanobacteria. J. Bacteriol. 130:82-91.
JOtJRNAL OF BACTERIOLOGY, Dec. 1977, p. 771-778 Copyright © 1977 American Society for Microbiology
Phycocyanin Synthesis and Degradation in the Blue-Green Bacterium Anacystis nidulans REGINALD H. LAU, MARGARET M. MAcKENZIE, AND W. FORD DOOLITTLE* Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7 Received for publication 16 August 1977
Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits; (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation. Blue-green bacteria owe their characteristic lans, a unicellular blue-green bacterium that colors to the biliproteins phycocyanin (PC) and contains only PC as the major biliprotein. Allen (in some species) phycoerythrin. These chro- and Smith (2) earlier characterized spectrophomophore-containing, accessory pigments cap- tometric changes under these conditions and ture much of the light energy essential for pho- concluded that nitrogen source (nitrate) starvatoautotrophic growth (4). At the same time, they tion results in loss of PC color (chlorosis), whereas restoration of nitrate promotes (after a can constitute up to 60% of the total cellular protein (4), and their syntheses must require a lag) rapid repigmentation, which must be comsignificant expenditure of that energy. It is pleted before cell growth can resume. Here, we clearly of selective advantage for blue-green bac- have asked (i) whether chlorosis indeed involves teria to control the rates of synthesis of PC and loss of PC apoprotein as well as loss of chromophycoerythrin apoproteins (and their cognate phore; (ii) whether de novo apoprotein synthesis chromophores) in ways that optimize, for any continues at a high relative rate during starvaenvironmental condition, functional phycobili- tion or is selectively depressed; (iii) whether the observed lag in repigmentation after nitrate resprotein content. Phycobiliprotein content, as measured spec- toration reflects a preferential lag in resumption trophotometrically, is indeed affected by (i) light of PC apoprotein synthesis; (iv) whether illumiquality (chromatic adaptation [3, 4, 14]), (ii) nation affects relative rates of PC apoprotein light quantity (9), and (iii) the availability of synthesis during repigmentation; and (v) whether C02, phosphate, and nitrate (2, 7, 8). However, the two PC apoprotein subunits (a, 16,000; /, spectrophotometric determinations reflect only 19,000 daltons [4]) are coordinately synthesized intact (chromophore-containing) PC content (or degraded) under these conditions. and do not differentiate changes effected at the MATERIALS AND METHODS level of apoprotein synthesis from those effected at the levels of protein turnover or chromophore Culture and labeling conditions. Wild-type A. attachment. Since it is commonly held (5, 6) nidulans was maintained as described previously (11, that blue-green bacteria are incapable of re- 12). One-liter cultures in the nitrate-containing mesponding to environmental variation by specific dium of Allen (1) were grown (except where noted) alterations in patterns of gene expression (pro- at 37°C under 5% C02-95% air in mechanically stirred Fernbach flasks that were 30 cm from four tein synthesis), it is especially important to char- 2.8-litercool-white fluorescent tubes. Cultures were laacterize, at the level of apoprotein formation, 40-W either with L-[4,5-3H (N)]leucine (New England beled molecular events during such pigmentation Nuclear Corp., Boston, Mass.) at 1 MCi and 0.01 I,g/ml changes. for two to four generations (10 to 20 h) or with L-[ UWe have chosen to examine alterations in 14C]leucine (New England Nuclear Corp) at 0.4 MCi intact-PC content, apoprotein content, and apo- and 0.3 Ag/ml for 20 min (pulse-labeling). Pigment and protein determination. For specprotein synthesis provoked by nitrogen source starvation and restoration in Anacystis nidu- trophometric determinations of PC, chlorophyll, and 771
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carotenoids, cells were harvested by centrifugation, frozen, and stored (I to 4 days) at -20'C. Chlorophyll and carotenoids were extracted from thawed cells with 2 to 5 ml of 80% acetone and measured at 665 and 460 nm, respectively (2). PC was determined in aqueous suspensions of acetone-extracted intact cells as described by Allen and Smith (2). All spectrophotometric values are expressed as the percentages of values obtained with samples taken at zero time (see Results). Protein content was determined as described by Ihlenfeldt and Gibson (8). Determination of 3H and '4C radioactivity in PC apoprotein: Method A. Samples were harvested by centrifugation, frozen for storage (1 to 4 days at -20'C), thawed, and suspended in :3.5 ml of lvsis buffer (11). Lysates obtained by passage through an Aminco French pressure cell at 1.1 x 10' kg/mi were freed from membranous debris bv cenitrifugation at 17,000 x g for 30 min (conditions that do not sediment biliproteins). Proteins were precipitated (30 min at room temperature) by the addition of trichloroacetic acid to 10%S, collected by centrifugation, washed twice with ethaniol, dried unlder vacuum, suspeended in sodium dodecyl sulfate (SDS)-containing sample buffer, heated to 100°C for 3 min, and loaded onto SDS-10% polyacrylamide gels, all as described previously (11). Molecular-weight-marker gels, loaded with phosphorylase a, bovine serum albumin, ovalbumin, pepsin, carbonic anhydrase, beta-lactoglobulin, cytochrome c, and/or purified A. nidulans PC (a generous gift of M. V. Laycock) were run simultaneously. For determinations of radioactivity, 1-mm gel slices were covered with 6 ml of toluene containing 57c P'rotosol (New England Nuclear Corp), 0.8K Omnifluor (New England Nuclear Corp.), and 0.3` water, shaken for 1 to 2 days at 37°C in polyethylene scintillation vials, and counted in a liquid scintillation counter programmed to correct for 4C crossover and quenchinig. Radioactivity in PC apoprotein a and /3 subunits and in total protein was quantitated by measuring areas under peaks on gel profiles (e.g., Fig. 5). Method B. For preparation of antisera, four rabbits were injected subcutaneously with approximately 1 mg of PC (showing only a and /3 subunit bands on SDS-polyacrylamide gels) in complete Freund adjuvant. Subsequent injections at 1, 2, 3, and 9 weeks were made in incomplete adjuvant, and rabbits were sacrificed bv heart puncture at 10 weeks. To 0.25-ml portions of cell lvsates prepared and free(d from membranous debris as described above, 24 ,ug of nonradioactive, purified PC (and, for experiments measuring PC apoprotein disappearance only, 12 ,l of a similarly prepared "standard lysate" of ["4C]leucine-labeled logarithmically grown cells) was added. Antiserum (0.25 ml) was then added, and the mixture was incubated for 20 min at 30°C and for 16 to 20 h at 4°C. Immunoprecipitates were collected by centrifugation at 27,000 x g for 5 min and washed twice with 5 ml of 0.05 M potassium phosphate (pH 7.0). Control experiments revealed that at least 90%/ of the added (nonradioactive) PC was precipitated under these conditions. Trichloroacetic acid precipitation, solubilization, gel electrophoresis, and determination of radioactivity in individual gel slices were performed as described above.
In experiments measuring disappearance of PC apoprotein (labeled with [rHlleucine before nitrate starvation), the ratio of 'H- to '4C-labeled, antibody-precipitated material migrating as PC was divided by the ratio of total "H- to 4C-labeled protein in the samples before immunoprecipitation (determined from profiles of S1)S-polyacrylamide gels of total soluble proteins). Since [ "'C]PC was a constant fraction of' the total "Clabeled (standard lysate) protein in all such mixtures, this procedure correcte(d for sample-to-sample differences in the efficiency of' cell lvsis, immunoprecipitate recovery, and gel loadiing. In experiments measuring the rate of PC apoprotein svnthesis after nitrate restoration (see Fig. 5 to , cells were labeled with [tH]leucine. starved, and then (at intervals after restoration of nitrate) labeled (2(1 min) with [14C]leucine. Relative incorporation of ['4Clleucine into PC apoprotein (corrected for variations in [r4Clleucine uptake, immunoprecipitation, and gel loading) was determined by dividing the ratio of 4C counts per minute to ~'H counts per minute in antibody-precipitated material migrating on gels as PC by the ratio 4C/ 'H in gels of total soluble protein. These relative values were multiplied by that fraction of total q'H counts per minute comprising PC in each sample (determined by method A). This last procedure yielded values for incorporation of '4C into PC apoprotein (as percent total 14C incorporation) and corrected for possible variations in relative [3H1PC content resulting from continued in vivo degradation of PC or turnover of other (non-PC) IH-labeled protein after nitrate restoration. Method B also permitted accuracy in determining 14C incorporation into PC in samples labeled early after nitrate restoration, when this protein comprised too small a fraction of total labeled material for determination by method A.
RE,SULTS Loss of PC apoprotein during nitrate starvation. Figure 1 illustrates results of a typical nitrate starvation-restoration experiment. Cells growing exponentially in nitrate-containing, [ XH]leucine-supplemented medium were washed and suspended at zero time in nitratefree (or, as a control, nitrate-containing) mediumn and incubated under growth conditions. Nitrate was restored to the starved culture at 63.5 h. At intervals during and after starvation, cells were harvested by centrifugation and frozen for subsequent spectrophotometric determination of chlorophyll, carotenoids, and intact (chromophore-containing) PC, and for independent determination of relative 3H-labeled PC apoprotein content. Turbidity at 750 nm was monitored in control and starved cultures throughout the experiment. Loss of spectrophotometrically measurable PC began soon after resuspension in nitrate-free medium, while turbidity, carotenoid, and chlorophyll levels continued to increase for at least 35 h (Fig. 1). (Continued increases in all four values were observed in the nitrate-containing
PHYCOCYANIN SYNTHESIS AND DEGRADATION
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FIG. 1. Spectrophotometric changes during nitrate starvation and after nitrate restoration. A logarithmically growing culture was washed and suspended (at zero time) in nitrate-free medium. Contents (per milliliter) of (0) PC, (A) chlorophyll, and (A) carotenoids, and turbidity (0) at 750 nm were followed. Nitrate was restored at 63.5 h (arrow). Inset shows results with a control (nitrate-containing) culture incubated in parallel. All values are expressed as percentages of values obtained at zero time.
control culture [inset] until this culture entered stationary phase at approximately 60 h.) These results are in qualitative agreement with those of Allen and Smith (2), although loss of PC absorbance was less rapid and complete than in their experiments. Loss of PC absorbance was paralleled by loss of apoprotein, as determined in two ways. In the first, method A, loss of 3H from PC (labeled before starvation) was quantitated by dividing 3H radioactivity in bands corresponding to PC apoprotein a and ,8 subunits by total radioactivity in all bands on 10% polyacrylamide gels of total soluble cellular proteins (for examples of such gels, see Fig. 5). Although other polypeptides must, of course, migrate with these subunits, the method can pretend to some accuracy, since (under present conditions) these two species alone comprise 10 to 20% of the total soluble protein. Values determined in this way are plotted (as a percentage of the values obtained immediately after nitrate removal) in Fig. 2.
773
In the second method (B), constant amounts of ['4C]leucine-labeled A. nidulans total soluble protein (prepared separately) and purified, carrier nonradioactive PC were added to portions of the same 3H-labeled (starved-cell) total protein preparations and precipitated with antiserum prepared in rabbits against purified A. nidulans PC. Immunoprecipitates were washed, solubilized, and resolved on 10% polyacrylamide gels. Gel profiles for samples taken at 0 and 63.5 h after starvation initiation are illustrated in Fig. 3. Values for immunoprecipitated 3H-labeled material migrating as PC on such gels were normalized (Materials and Methods) and are plotted (as percentages of values obtained immediately after nitrate removal) in Fig. 2. Rates of PC apoprotein disappearance derived by these two independent methods are in reasonable agreement with each other and with the rate of loss of spectrophotometrically measurable (chromophore-containing) PC. We conclude that chlorosis indeed involves apoprotein degradation as well as chromophore loss, although it is not possible to say (because of scatter in the data) how precisely these events are coordinated. It is possible to compare individual rates of loss of the two subunits of PC apoprotein, since these were at least partially resolved on SDS-polyacrylamide gels of immunoprecipitated material. Values of 3H counts per minute/14C counts per minute for , (peak slice) divided by 3H counts per minute/14C counts per
HOURS STARVATION
FIG. 2. Correlation of loss of spectrophotometrically measurable PC with loss of PC apoprotein subunits. Relative PC apoprotein content determined by SDS-gel electrophoresis of total soluble proteins (0, method A) or of proteins precipitated by antiserum directed against purified PC (0, method B). (O) Spectrophotometrically measurable PC (from Fig. 1). Values are expressed as percentages of values obtained at zero time. Inset: Ratio of PC a and ft subunits during starvation.
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LAU, MACKENZIE, AND DOOLITTLE
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minute for a (peak slice) are shown in Fig. 2 and indicate that degradation of the two subunits is coordinate. De novo synthesis of PC during nitrate starvation. Rates of de novo synthesis of PC apoprotein during nitrate starvation were determined in an experiment (similar to that illustrated in Fig. 1) in which [3H]leucine-labeled, washed, and nitrate-starved cultures were pulselabeled with ['4C]leucine at 0, 3, 13, and 37 h after starvation initiation. After pulse-labeling (20 min), cells were harvested and lysed. Relative rates of PC apoprotein synthesis were taken as fractions of total 14C radioactivity migrating as a and subunits on polyacrylamide gels of total soluble protein. Although substantial incorporation of 14C into total protein was observed during nitrate starvation, in no sample (including that pulse-labeled immediately after starvation) did material migrating as PC comprise more than 1% of the total '4C-labeled material. (By contrast, up to 8% of the "4C-labeled protein from cultures pulse-labeled 10 h after nitrate restoration migrated as PC [see Fig. 7]). SDS-gel electrophoresis of immunoprecipitated samples also indicated that no higher-molecularweight (potential precursor) polypeptides with PC antigenic determinants were labeled during nitrate starvation. Thus, starvation either results in rapid preferential degradation of newly
synthesized PC apoprotein subunits or (as we feel more likely) specifically depresses expression of PC apoprotein structural genes. Resynthesis of PC after restoration of nitrate. Experiments such as that illustrated in Fig. 1 (and the earlier data of Allen and Smith [2]) suggest that, although PC levels (measured spectrophotometrically) begin to increase soon after readdition of nitrate, maximal rates of pigment accumulation are not achieved for several hours. A similar delay in the resumption of high rates of accumulation of spectrophotometrically measurable PC is seen in Fig. 4, which also illustrates the effects of (i) exclusion of light, addition of 3-(3,4-dichlorophenyl)-1,1-di(ii) methylurea (an inhibitor of photosystem II), and (iii) addition of chloramphenicol, all immediately before nitrate restoration. This delay could result from a lag in resumption of chromophore synthesis (with consequent accumulation of free apoprotein), a lag in resumption of protein synthesis affecting all cellular components, or a specific lag in the resumption of PC apoprotein synthesis. Similarly, the failure of unilluminated (or 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated) cells to accumulate spectrophotometrically measurable PC rapidly, even long after nitrate restoration, could reflect the overall depression of protein synthesis previously observed in darkened cells (11) or a specific effect
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proteins (see Materials and Methods). In samples from the illuminated culture pulselabeled soon after nitrate restoration, PC a and ,B subunits comprised a small fraction of total "C-labeled proteins and polypeptides of molec-7 ular weights between 20,000 and 80,000 (some, 0 but not all, of which comigrated with 3H-labeled E species) predominated (Fig. 5). ImmunoprecipiCL4 tated [14C]PC was similarly low (Fig. 6), and no higher-molecular-weight proteins (potential precursors) carrying PC antigenic determinants were detectable on gels of immunoprecipitates. 5 10 Percentages of "C-pulse-labeled material from illuminated and darkened cells precipitated HOURS AFTER NITRATE RESITRAIIUN FIG. 4. Resumption of accumulation of spectro- by anti-PC and migrating as PC on SDS-polyphotometrically measurable PC after restoration of acrylamide gels (method B, normalized as denitrate to a starved culture. A logarithmically grow- scribed in Materials and Methods) are plotted ing culture was washed, suspended in nitrate-free against time after nitrate restoration in Fig. 7. medium, and incubated until spectrophotometrically Values determined by method A are also shown measurable PC fell to about one-half of its original (for the illuminated culture only). Agreement value. At zero time, the culture was divided into four between values determined by these two indesubcultures, nitrate was restored to each, and PC pendent methods is good, although quantitation content was determined spectrophotometrically at intervals thereafter. Symbols: 0, control (illuminated) of areas under the peaks is obviously uncertain subculture; 0, unilluminated subculture; V, subcul- when relative PC apoprotein synthesis is low (e.g., panels A and B of Fig. 5), and we consider ture containing 100 ug of chloramphenicol per ml; A, subculture containing 10 4ug of 3-(3,4-dichloro- method B to be the more accurate. Figure 7 phenyl)-1,1-dimethylurea per ml. All values are ex- also presents, for both cultures, results of specpressed as percentages of values at the time of nitrate trophotometric measurements of intact (chrorestoration. mophore-containing) PC per milliliter of culture and determinations of total protein per milliliter on relative rates of chromophore and/or apopro(both presented as the percentages of values tein formation. To distinguish between these possibilities, ex- before nitrate removal). It should be realized periments like those illustrated in Fig. 5 to 7 that these latter are measures of total pigment were performed. [3H]leucine-labeled cells were and protein accumulation, whereas measurewashed and starved for nitrate, as before. After ments of percent "C incorporation into PC respectrophotometrically measurable PC levels flect relative rates of apoprotein synthesis. In illuminated cells, relative rates of PC apofell to about one-half their original value, nitrate protein synthesis rose 6- to 10-fold during the was restored (zero time), and the culture was divided into two subcultures, one maintained first 9 h after nitrate restoration, becoming conunder continuous illumination and the other stant (at about 8% of the total-soluble-protein maintained in darkness. At intervals, samples synthesis) shortly after the resumption of accumulation of spectrophotometrically measurable were removed and incubated (under the same illumination conditions) with ['4C]leucine for 20 PC and total protein. Comparisons of 14C/3H min before harvesting for lysis and protein ex- ratios in resolved a- and,8-subunit peaks on gels traction. Incorporation of ['4C]leucine into PC of immunoprecipitates revealed that the synthesis rates of the two PC apoprotein subunits are was determined as a fraction of the total incorporation by the two methods described above. coordinately controlled under these condiRepresentative profiles of 10% polyacrylamide tions. We cannot readily explain the failure of gels of total soluble proteins from samples taken relative PC apoprotein synthesis to reach levels at 1, 3, 9.5, and 15.5 h after nitrate addition comparable to those observed for PC apoprotein (illuminated culture) are shown in Fig. 5. Profiles content (ca. 20%, by method A) in steady-stateof radioactivity in gels of proteins precipitated labeled exponentially growing cultures. It may from these same samples with anti-PC are be relevant that the illuminated culture was shown in Fig. 6. For these latter, 3H radioactivity entering the stationary phase towards the end of the experiment illustrated in Fig. 5 to 7 and was normalized to constant recovery (relative to the 1-h sample) of [3H]PC a and ,f subunits; that, in other experiments (e.g., illustrated in '4C values were multiplied by the same factor Fig. 1), PC apoprotein synthesis rose from 2 to and corrected for differences in total 14C labeling 12% of the total protein synthesis during the by using values for total "4C and 3H in soluble first 10 h after nitrate restoration. Similarly, we :n
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before nitrate starvation and pulse-labeled with l4 C]leucine after nitrate restoration. An exponentially growing culture was labeled with ['Hjleucine for several generations, washed, and suspended in nonradioactive, nitrate-free medium. Nitrate was restored after spectrophotometrically measurable PC had fallen to about one-half of its original level, and incubation (under illumination) was continued for 17.5 h. At intervals. samples were withdrawn and pulse-labeled (20 min under illuminati'on) with [l4C/leucine. Cells were harvested, and the distribution of 'H and 14C radi'oactivities in total soluble proteins was analyzed as described in the text. Results are shown for samples pulse- labeled at I (A), 3 (B), 9.5 (C), and 15.5 (D) h after nitrate restorati'on. Numbers in panel (A) indicate molecular weights (xIO') as determined by compari'son with simultaneously run gels containing "marker" proteins of known molecular weights. Positions of phycocyanin apoprotetin a and fl subunits are i'ndicated. Symbols: O, 'H counts per minute; *, 14C counts per minute.
776
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FIG. 7. Relative rates of synthesis of PC apoprotein and absolute levels of spectrophotometrically measurable PC and total protein after nitrate restoration. Relative rates of PC apoprotein synthesis (i.e., percentage of total 14C-pulse-labeled soluble protein comprising PC a and ,B subunits) were determined by methods A (0) and B (0), as described in the text, and are plotted against time of initiation of pulse-labeling. Spectrophotometrically measurable PC (O) and protein (V) content (per milliliter of culture) were determined with portions of the same samples and are expressed as percentages of values determined immediately after the initial removal of nitrate. (A) Culture continuously illuminated; (B) culture darkened upon restoration of nitrate.
cannot exclude the possibility that PC apoprotein is considerably more stable than the bulk of polypeptides synthesized soon after nitrate restoration. This possibility cannot be tested
directly, since pulse-labels can only slowly be "chased" from internal pools by the addition of excess nonradioactive amino acid. However, 3H label (which was adequately chased in these experiments) was redistributed (resulting in a 50% increase in relative [3H]PC content) after nitrate restoration, thus indicating that some (non-PC) proteins are degraded during the recovery period. In unilluminated cells, PC apoprotein synthesis after nitrate restoration follows a complex course (Fig. 6). Early after restoration this protein constituted, in darkened cells, a larger fraction of total pulse-labeled material than in ffiluminated cells. The failure of darkened cells to accumulate spectrophotometrically measurable PC thus cannot be attributed to lowered expression of PC apoprotein structural genes and can be adequately explained by the overall depression in protein synthesis reported previously (11) and apparent in Fig. 7. DISCUSSION We have confirmed a proposal (often an implicit assumption) made in earlier work dealing with pigmentation changes in blue-green bacteria: that spectrophotometric determinations of PC reflect (at least in the case of nitrate starvation and restoration) events at the level of apoprotein degradation and synthesis. (Bennett and Bogorad have provided similar evidence for
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LAU, MACKENZIE, AND DOOLITTLE
changes in relative rates of PC and phycoerythrin apoprotein synthesis during chromatic adaption in Fremyella diplosiphon [3].) It seemed to us particularly important to provide such confirmation for nitrate starvation-induced chlorosis, since blue-green bacteria do possess other nitrogen stores (cyanophycin granules [10]) and the wholesale dismantling of the lightharvesting apparatus may seem an inappropriate initial response to nitrogen limitation. We can now conclude that (i) nitrate starvation-induced chlorosis is accompanied by coordinate loss of the two PC apoprotein subunits; (ii) during starvation, de novo PC apoprotein synthesis is specifically depressed; (iii) this specific depression persists for several hours after nitrate is restored (a- and /3-subunit syntheses recover in a coordinate fashion and reach maximal rates at about 10 h, shortly after the resumption of accumulation of total protein and spectrophotometrically measurable PC); and (iv) both rates of overall protein formation and relative rates of PC apoprotein synthesis are affected by light. These results should be viewed in the context of the hypothesis, put forward by Carr and his collaborators (5, 6), that blue-green bacteria are deficient in controls exerted at the level of gene expression and that the relative inability of these organisms to use potentially metabolizable exogenous sources of carbon and energy other than CO2 and light stems from this deficiency. It indeed appears that exogenous metabolites which are not normally required for (and which do not normally greatly stimulate) photoautotrophic growth do not evoke changes in specific activities of enzymes involved in their metabolism (5, 6, 12). However, environmental stimuli to which the growth of these organisms does respond (light, C02, phosphate, and nitrate) markedly influence patterns of protein synthesis (8, 11, 13), and it is simplest to attribute these specific environmental effects to controls operating at the level of gene expression.
J. BACTERIOL. ACKNOWLEDGMENTS This work was supported by grant MT 4467 from the Medical Research Council of Canada. We are very grateful to M. V. Laycock for samples of purified phycocyanin and to R. A. Singer for helpful discussions.
LITERATURE CITED 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 2. Allen, M. M., and A. J. Smith. 1969. Nitrogen chlorosis in blue-green algae. Arch. Mikrobiol. 69:114-120. 3. Bennett, A., and L. Bogorad. 1973. Complementary chromatic adaptation in a filamentous blue-green alga. J. Cell Biol. 58:419-435. 4. Bogorad, L. 1975. Phycobiliprotein and complementary chromatic adaptation. Annu. Rev. Plant Physiol. 26: 369-401. 5. Carr, N. G. 1973. Metabolic control and autotrophic physiology, p. 39-65. In N. G. Carr and B. A. Whitton (ed.), The biology of the blue-green algae. Blackwell Scientific Publications, Oxford. 6. Delaney, S. F., A. Dickson, and N. G. Carr. 1973. The control of homoserine-O-transsuccinylase in a methionine requiring mutant of the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 79:89-94. 7. Eley, J. H. 1971. Effect of carbon dioxide concentration in the blue-green alga Anacystis nidulans. Plant Cell Physiol. 12:311-316. 8. Ihlenfeldt, M. J. A., and J. Gibson. 1975. Phosphate utilization and alkaline phosphatase activity in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102: 22-28. 9. Jones, L. W., and J. Myers. 1965. Pigment variations in Anacystis nidulans induced by light of selected wavelengths. J. Phycol. 1:7-14. 10. Simon, R. D. 1973. Measurement of cyanophycin granule polypeptide content in the blue-green alga Anabaena cylindrica. J. Bacteriol. 114:1213-1216. 11. Singer, R. A., and W. F. Doolittle. 1975. Control of gene expression in blue-green algae. Nature (London) 253:650-651. 12. Singer, R. A., and W. F. Doolittle. 1975. Leucine biosynthesis in the blue-green bacterium Anacystits nidulans. J. Bacteriol. 124:810-814. 13. Stevens, S. E., Jr., and C. Van Baalen. 1974. Control of nitrate reductase in a blue-green alga: the effects of inhibitors, blue-light, and ammonia. Arch. Biochem. Biophys. 161:146-152. 14. Tandeau de Marsac, N. 1976. Occurrence and nature of. chromatic adaptation in cyanobacteria. J. Bacteriol. 130:82-91.
