Alterations in cell pigmentation, protein expression

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Within the range of iron (4.2 x 10-5 - 5.1 x 10-9 M FeCl?), growth rates were not ... l'induction de ces prottines et la production de sidCrophores extracellulaires.
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Alterations in cell pigmentation, protein expression, and photosynthetic capacity of the cyanobacterium Oscillatoria tenuis grown under low iron conditions Charles G. Trick, Steven W. Wilhelm, and Christopher M. Brown

Abstract: To better describe the iron-limited nutrient status of aquatic photosynthetic microorganisms, we examined the effects of iron limitation on pigment content, maximum rates of photosynthetic oxygen evolution, and respiratory oxygen consumption in the filamentous cyanobacterium Oscillatoria tenuis Ag. Within the range of iron (4.2 x 10-5 - 5.1 x 10-9 M FeCl?), growth rates were not limited by photosynthetic capacity but rather by another, as of yet undetermined, iron-requiring cellular function. We have also investigated membrane proteins that are induced when the cells are grown in low iron medium. Using membrane fractionation techniques we were able to recognize specific proteins localized in the outer membrane and periplasmic space of 0. tenuis. The recovery of growth rates at low iron levels occurred in parallel with the induction of these proteins and the production of extracellular siderophores. The additional iron acquired by this high affinity transport system did not reestablish photosynthesis in 0. tenuis to the iron-satiated level but did reestablish growth to iron-replete levels. Oscillatoria tenuis appears to invoke an alternate physiology to compensate for iron deficiency. Key words: cyanobacteria, iron, Oscillatoria tenuis, periplasmic proteins, photosynthesis.

Resume : Dans le but de mieux dCcrire 1'Ctat nutritif limit6 en fer des microorganismes aquatiques photosynthCtiques, la prCsente Ctude a considCr6 les effets de la limitation en fer sur la teneur en pigments, les taux maximums d'Cvolution de l'oxygbne photosynthCtique et la consommation respiratoire de l'oxygbne chez la cyanobactCrie filamenteuse Oscillatoria tenuis Ag. A l'inttrieur d'une gamme de concentrations en fer (4,2 x - 5,l x 10-9 M de FeCI3), les taux de croissance n'ont pas CtC limit& par la capacitC photosynthttique mais plut6t par une autre fonction cellulaire non encore dCterminCe requCrant la prCsence de fer. L'Ctude a Cgalement port6 sur les protkines membranaires qui sont induites au cours de la croissance des cellules sur un milieu faible en fer. Les techniques de fractionnement membranaire ont rendu possible la reconnaissance des protkines sptcifiques IocalisCes dans la membrane externe et dans l'espace pCriplasmique des cellules d'O. tenuis. Le recouvrement des taux de croissance B de faibles niveaux de fer est survenu en parallble avec l'induction de ces prottines et la production de sidCrophores extracellulaires. Le fer additionnel acquis par ce systbme de transport d'affinitk ClevC n'a pas rCtabli la photosyntMse chez 1'0. tenuis au niveau de la satiCtC en fer, mais il a rCtabli la croissance en prksence de niveaux saturCs en fer. L'O. tenuis semble recourir a une physiologie alternative pour compenser une dCficience en fer. Mots clks : cyanobactCrie, fer, Oscillatoria tenuis protkines pCriplasmiques, photosynthbse. [Traduit par la rCdaction]

The influence of iron on the growth and physiology of microbial primary producers has attracted considerable attention in recent years (Martin et al. 1990, 1991, 1994). For prokaryotic Received March 22, 1995.Revision received September 7, 1995.Accepted September 8,1995.

C.G. Wick,' S.W. Wilhelm, and C.M. Brown. Department of Plant Sciences, The University of Western Ontario, London, ON N6A 5B7, Canada. Author from whom reprints should be requested. Present address: Marine Science Institute, The University of Texas at Austin, P.O. Box 1267, Port Aransas, TX 78373, U.S.A. Can. J. Microbiol. 41: 1117-1123 (1995). Printed in Canada / ImprimC au Canada

primary producers, iron is required in cellular processes such as electron transport, porphyrin synthesis, and nitrogen metabolism (Peschek 1979; Boyer et al. 1987), or as a regulatory agent for functions under the control of iron availability (Laudenbach et al. 1988). Of particular interest is the effect of iron availability on filamentous species of cyanobacteria due to their role in the introduction of nitrogen into aquatic systems via N2fixation (Rueter et al. 1990; Paerl et al. 1994).While the marine Trichodesmium spp. remain difficult to culture, we can successfully maintain the freshwater congere Oscillatoria tenuis Ag. Iron limitation in cyanobacteria results in a decrease in both cell phycobiliproteinlevels (Guikema and Sherman 1983;

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Sandmann 1985) and rates of chlorophyll a biosynthesis (Oquist 1974; Sandmann 1985; Wilhelm and Trick 199%). Peschek (1979) found that the maximum photosynthetic rate of Anacystis nidulans R2 was maintained at approximately 200 pmol 02.h-l. mg chlorophyll a-l, irrespective of iron limitation. However, Sandmann (1985) found that Aphanocapsa sp. exhibited lower rates of both photosynthesis and respiration under iron limitation and concluded that photosynthesis was limited not by chlorophyll a content but by electron transport. In each instance, iron-limited cyanobacterial cells display lower chlorophyll a content and a decrease in the phycocyanin/chlorophyllratio (Oquist 1974; Guikema and Sherman 1983; Sandmann 1985), which result in lower rates of photosynthetic oxygen evolution on a per cell basis. Paerl et al. (1994) have demonstrated in natural populations of Trichodesmium spp. that iron availability may limit carbon fixation, chlorophyll a levels, and nitrogenase activity. However, limited conclusions have been drawn with respect to the effects of iron limitation on physiological parameters, such as light-limited photosynthetic rates and light compensation points of cyanobacterial cultures. In this paper we consider the influence of the level of external iron on photosynthetic rates and pigmentation levels for the filamentous cyanobacterium 0 . tenuis Ag. This strain was chosen because it can successfully grow over a wide range of iron concentrations by producing iron-chelating siderophores (Brown and Trick 1992). To satiate the iron demands of cyanobacteria from low iron environments, a high affinity iron transport system, similar to the transport systems in other bacteria, may be induced. In cyanobacteria, evidence for the ironregulated release of extracellular siderophores has been presented for several species (Boyer et al. 1987; Keny et al. 1988; Wilhelm and Trick 1994); the corresponding the transport protein(s) specific to iron-siderophore have yet to be identified. Although there is no direct evidence for iron-siderophore receptor proteins in cyanobacteria, low iron enhanced proteins have been identified. Iron-regulated changes in the outer membrane polypeptide profiles of several cyanobacteria have been documented (Scanlan et al. 1989,Wilhelm 1994). In this paper, we provide evidence for low iron inducible membrane proteins from the freshwater filamentous cyanobacterium 0 . tenuis. We demonstrate that the production of the membrane proteins occurs at iron levels that also regulate the production of the extracellular siderophores. Do cells that obtain iron from low iron environments through the activities of an iron-siderophore scavenging system share the same physiological status as cells that are iron satiated by inorganic iron transport? Oscillatoria tenuis provides a unique example, since the growth rates of the organism are identical under the two different states of iron availability.

Materials and methods Oscillatoria tenuis Ag. was obtained from the Algal Culture Collection at The University of Texas at Austin (UTEX 428) and rendered axenic by physical manipulation (Brown 1991). Cultures (50 mL in 250-mL Erlenmeyer flasks) were grown at 25°C under a light flux of 70-100 pmol photons. m-2. s-I on an orbital shaker.

BG-11 media (Rippka et al. 1979)was modified by replacing fenic ammonium citrate with FeC13.6H20 (Keny et al. 1988; Brown and Trick 1992).Tris-HC1 was added to a final concentration of 0.5 g . L-I to maintain the pH of the media at 7.4. All water and nutrient stocks (except the trace metal and vitamin stocks) were passed through a Chelex-100 resin column to remove residual iron (Price et al. 1989). The range of iron concentrations used was from 4.2 x to 5.1 x 10-9 M FeC13.6H20, with each iron concentration differing by approximately one order of magnitude from the next. For all experiments, cells were preconditioned to the experimental iron concentration for a minimum of 10 transfers to ensure that cellular storage of iron did not influence the experiment. Cells were harvested in late exponential growth for all experiments. Cell enumeration was completed as previously described (Brown and Trick 1992).

Measurement of pigments Extractable cell chlorophyll a concentrations were obtained by collecting cells on glass fiber filters (Whatman GF/C). The filters and cells were ground with a mortar and pestle in 90% acetone, and extracts were placed in the dark at room temperature for 24 h. One drop of a saturated MgC03 solution was added prior to incubation to buffer the extracts. Absorbances of acetone extracts were obtained at 663,645, and 630 nm, and chlorophyll a values were determined using the empirical formula of Richards and Thompson (1952). Phycocyanin/cell and phyocyanin/chlorophy11a ratios were estimated using wholecell spectra as described by Myers et al. (1980). Measurement of photosynthesis and respiration Photosynthesis was measured using a Clark-type O2 electrode (Hansatech, Ltd., Norfolk, U.K.) at a constant temperature of 20°C (Wilhelm and Trick 1995~). Light-limited rates of photosynthetic 0 2 evolution were obtained at 18 different light levels ranging from 0 to 1000 pmol photons. m-2. s-I photosynthetically active radiation (PAR). A series of photodiodes, model LH 36 UB (Hansatech Ltd., A,, = 660 nm), provided the light source to illuminate the cells at each photon fluence rate. Photosynthetic rates were calculated at the end of each interval to generate photosynthesis versus irradiance (P vs. I ) curves. Values for apparent quantum yields (a)were derived using the slopes of the light-limited regions of the P vs. I curves and expressed as pmol 0 2 . cell-' .s-I divided by pmol photons. m-2. s-l, as well as normalized to pg chorophyll a. Light compensation points were obtained from the x-axis intercepts of these plots and expressed as pmol photons. m-2. s-I (PAR). Respiration rates were obtained by measuring oxygen consumption in the dark. Results are presented (+SE) for four independant cultures for all experiments. Preparation of outer membranes Outer membrane fractions were isolated by sarkosyl solubilization of the cytoplasmic and thylakoid components as described in Wilhelm and Trick (1995b), with the exception that cells were disrupted with 0.5-mm glass beads (Biospec Products). The mechanism of sarkosyl action has been described elsewhere (Nikaido 1994). To release proteins from the periplasmic space, cells were osmotically shocked by the procedure of Neu and Heppel

Trick et al. Table 1. Effect of iron concentration on 0.tenuis growth rates and apparent quantum yield (f SD). -

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Total iron

Growth rate (day-')

Fig. 1. Whole-cell spectra of 0.ten14i s cells gown in medium containing high levels of added iron (4.2 x 10-" FeCI3) M FeCl?) (-Fe). (+Fe)and low levels of added iron (5.1 x

ci values

Chlorophyll

Cell

Note: Values for a are normalized to both per chlorophyll (prnol 02.p g chlorophyll a-I . s-' divided by pniol photons . m-2. s-') and per cell (pmol O2 . cell-' . s-I divided by kmol photons . m-2 . s-').

(1965). Cells from 1 L of late exponential culture were collected by filtration onto glass fiber filters (Whatman GFIC) and quickly resuspended in 20 mL of 20% sucrose in 0.03 M Tris (pH 8.0) at 24°C. Na2EDTAwas added to a final concentration of 1 mM and the suspensions were shaken gently for 10 min. Cells were recollected on glass fiber filters and osmotically shocked by quick resuspension in 20 mL of cold (4°C) distilled water. Cells were removed by filtration through a 1-pm membrane filter and the cell-free filtrate was lyophilized to aprotein concentration of greater than 1 pg .mL-1. The complete absence of phycocyanin in the periplasrnic protein fraction (determined visually) indicated that minimal lysis of cells occurred during the osmotic shock. 14C radiolabelling of membrane proteins To investigate de novo synthesis of membrane proteins, cells grown in medium containing 4.2 x 10-5 M FeC13 were collected on glass fiber filters. The concentrated cells were equally distributed between flasks containing 20 mL of medium enriched with either 4 pM ferric chloride or 4 pM femc plus 50 pg . mL-1 of EDDA (ethylenediamine di(hydrox1)phenylacetic acid, a commercially available iron chelator). To each flask, 6 x 107 cpm of Na2H14CO3 was added. Flasks were incubated at 25°C and 70 pmol phot0n.m-2,s-I for 24 h. Cells were collected onto glass filters and membrane proteins were separated and visualized as described previously.

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) Standard SDS-PAGE was performed using the buffer system of Laemmli (1970). Gradient gels were established with a gradient from 15 to 7.5% acrylamide. Proteins were visualized using the staining technique of Merril et al. (1981). Fluorograms were prepared with an initial series of three 30-min equilibrium washes in 100 mL of dirnethyl sulfbxide (DMSO), followed by a 3-h incubation in 20 g PPO (2.5-diphenyloxazole) in 100 mL DMSO. Gels were transferred for a 12-h water rehydration step, after which the gels were dried and exposed to photographic X-ray film at -70°C.

Results This set of experiments was designed to determine if physiological changes occurred in this cyanobacterium as compensa-

Wavelength, nm

tion for low iron availability. Growth rates remained constant at the highest levels of added iron (Table 1). The growth rate of 0. tenius did decline significantly as iron was decreased to 3.1 x 10-8 M. The highest growth rate was seen at the lowest level of total iron (5.1 x 10-9 M), although this value was not statistically different from the growth rate at the highest iron level (P < 0.005). At the highest and lowest added iron concentrations (4.2 x 10-5 and 5.1 x 10-9 M FeC13 respectively), pigment composition was altered. The absorption peak corresponding to the chlorophyll a maxima (678 nm) was reduced in the whole cell spectra (Fig. 1). The 620-nm peak that corresponds to the presence of the accessory pigment phycocyanin (PC) remained the same. Extractable pigments from 0. tenuis cells cultured in BG-11 media containing 4.7 x 10-6 M FeC13had the highest chlorophyll a content (Fig. 2A). Cultures grown in the lowest iron concentration (5.1 x 10-9 M FeC13)contained only 10% of this amount. Surprisingly, cells cultured at the highest concentration of added iron (4.2 x 10-5 M FeC13)had a slightly lower chlorophyll a content than those grown at the lower iron concentration of 4.7 x 10-6 M FeC13. The sharpest decreases in chlorophyll a content of 3- and 2.3-fold occurred between FeC13 concentrations of 4.7 x 10-6 and 4.1 x 10-7 M and between 3.1 x 10-8 and 5.1 x 10-9 M, respectively. PC concentrations were estimated from whole-cell spectra of high and low iron cultures of 0. tenuis (Fig. 2B). The concentration of PC decreased from 0.0456 0.0119 pg . cell-1 when cells were cultured with 4.2 x 10-5 M FeC13 to 0.0191 f 0.0035 pg . cell-' at 5.1 x 10-9 M iron. While the PC content followed the decline in chlorophyll a content between FeC13 concentrations of 4.2 x 10-5 and 3.1 x 10-8 M relative to chlorophyll a, PC concentrations stabilized at 3.1 x 10-8 M FeC13, while chlorophyll a per cell continued to be reduced (Fig. 2A). The PC/chlorophyll a (A 625/A678)ratio in whole cell spectra of 0.tenuis increased when cells were grown under low iron conditions (Fig. 2C). A gradual increase was seen between iron concentrations of 4.2 x 10-5 FeC13 and 3.1 x 10-8 M FeCI3, while a stronger increase accompanied a decrease in the iron concentration from 3.1 x 10-8 to 5.1 x 10-9 M FeC13.

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Fig. 2. Pi-gent levels and photosynthetic parameters for 0.tenuis cells grown in medium containing .specific concentrations of added iron. (A) Cellular chlorophyll a; (B) cellular phycqnnin: (C) phycocyanin/chlorophyll a ratio: (D) light-saturated photosynthetic rates: (E)respiration rates; IF)light com~nsationpoints n = 4. Chl a, chlorophyll a.

Iron, M FeC13

Light-saturated rates of photosynthetic oxygen evolution decreased from 3.10 f 0.484 ymol 0 2 . 1O8cells-' .h-1 in high iron medium to 0.61 f 0.049 pmol 0 2 . 108 cells-'. h-1 in the medium with the lowest amount of added FeC13 (Fig. 2D). M FeC13,however, Cells grown in media containing 4.7 x had the highest photosynthetic rate. The changes in these rates followed the same general pattern as the changes in cellular chlorophyll a content outlined above, suggesting that chlorophyll a content is a major determinant of the cellular photosynthetic rate for the range of iron concentrations examined. There was no significant change (P < 0.05) in the photosynthetic rates for these cultures when normalized to chlorophylla

Iron, M FeC13

over the range of iron concentrations (Brown 1991). Apparent quantum yields, calculated on a per chlorophyll a basis, remained relatively constant over the range of iron availabilities (Table I). Determination of a on a per cell basis, however, demonstrated an order of magnitude difference from the highest a (at 4.7 x M FeCI3) to the lowest a (at 5.1 x 10-9 M FeCI3). Cellular rates of respiration displayed a net decrease over the range of iron concentrations tested (Fig. 2E). The rate initially increased as the iron concentration was decreased to 4.7 x M FeCI3,but a significantdecline in the rate of respiration accompanied a decrease in the iron concentration to 5.1 x

Trick et al.

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Fig. 3. (A) Silver-stained SDS-polyacrylamide gel of outer membranes from 0.tenuis cells cultured at specific concentrations of iron. Lane 1,4.2 x 10 - 5 M FeC13; lane 2, 5.1 x 10 -9 M FeCI3. (B) Fluorogram of I4C-labelled outer membrane proteins of 0. tenuis. Lane 1, culture labelled with 14Cduring incubation with 4.0 x 10-6 M FeC13; lane 2, culture perturbed with 14Cwith 4.0 x lop6M FeC13 plus 50 pg EDDA . mL-'. Molecular masses in kDa are indicated along the sides of the gels.

Fig. 4. Silver-stained SDS-polyacrylamide gel of periplasmic (osmotic shock released) proteins from 0. tenuis cells cultured at specific concentrations of iron. Lane 1,4.2 x 10 - 5 M FeC13; lane 2,4.7 x 10-6 M FeC13; lane 3,3.1 x M FeC13; lane 4, 5.1 x 10 -9 M FeC13. Molecular masses in kDa are indicated along the sides of the gel.

High Fe 10-9 M FeC13. The light compensation point of 0. tenuis increased continuously from high to low iron concentrations (Fig. 2F). The overall increase in the light compensation point was significant (P < 0.05). The outer membrane fraction from 0. tenuis had a distinct orange color that, when spectrally examined, provided absorbance peaks at 464,489, and 525 nm (see Omata and Murata 1983,1984). Solubilization of this fraction, followed by protein separation by SDS-PAGE, allowed for the identification of proteins more abundant in outer membranes of cells grown in low iron medium (Fig. 3A). The most obvious were two proteins with relative molecular masses of 53 and 5 1 kDa. Several less abundant iron-regulated proteins were also identified. However, the iron-regulated induction of these proteins could not be determined unambiguously. To identify low iron regulated outer membrane proteins, Na2H14C03 was used to label the outer membranes of iron-satiated cells in comparison with the de novo profile of cells introduced into a low iron environment (EDDA added). A fluorogram of the solubilized and separated outer membrane proteins revealed the accumulation of the label in several proteins in the outer membranes of the low iron grown cells (Fig. 3B, lane 2). Three of these proteins, having relative molecular masses of 158, 81, and 53 kDa, were strongly labelled. While a 53-kDa band was also detected in the ironreplete sample, there appeared to consistently be a lower expression in this fraction. Two other proteins of 200 and

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Low Fe

5 1 kDa were less strongly labelled, although both were easily detected above the background label. Outer membranes from the iron sufficient cells (Fig. 3B, lane 1) contained a large number of weakly labelled proteins. A large number of proteins were released from 0. tenuis by osmotic shock (Fig. 4). Periplasmic proteins were isolated from cells grown in arange of iron concentrations,from 4.2 x to 5.1 x 10-9 M. Only a single iron-regulated protein was identified in the periplasmic fraction. This protein, which has a relative molecular mass of 56 kDa, was not easily detected owing to its close proximity to a more abundant protein of approximately 55 kDa. There were also a number of other proteins whose expression appears to be reduced in the low iron samples.

Discussion Cellular chlorophyll a and PC contents were the most useful indicators of iron availability in 0. tenuis. Both values declined significantly as the amount of iron available in the medium decreased. The increase in the PC/chlorophyll a ratio might be expected to affect a owing to the larger relative number of phycobilisomes available for light harvesting. However, the a (normalized to chlorophyll a ) remained relatively constant, suggesting that the excess PC may be maintained in the cell in a nonfunctional or less efficient state. This functional

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uncoupling of the phycobilisomes from the chlorophyll a reaction cores has been reported in photobleached or photoinhibited cells of Anabaena variabilis (Nultsch et al. 1990). Oscillatoria tenuis displayed lower rates of respiration per cell under low iron growth. A decrease in the rate of respiration of other cyanobacteria has been attributed to the loss of two key enzymes, cytochrome oxidase and NAD(P)H oxidase (Peschek 1979; Sandmann 1985). Similar reductions in these enzymes may occur in 0. tenuis. However, in 0. tenuis, the observation that respiration was not as strongly affected as photosynthesis reflects the higher iron demands of photosynthetic electron transport as compared with those of respiratory electron transport. The increased ratio of respiration to photosynthesis in iron-limited cells resulted in higher light compensation points. Iron-deprived cells, therefore, require higher photon fluence rates to achieve a net oxygen production. It is conceivable that, at the photon fluence rates at which 0. tenuis is cultured (76-100 kmol. m-2 - s-I), photosynthesis by these low iron cultures barely compensates for respiration. The intensity of light required for cells to obtain maximum rates of photosynthesis was beyond that encountered by the cells in culture, so that the actual difference in the gross photosynthetic rate between high and low iron grown cells at 70 kmol. m-2. s-I of light was probably less than that observed at 200 pmo1.m-2. s-I of light. In spite of the changes in the pigmentation and photosynthetic capacity of low iron grown 0. tenuis, the growth rate of cells was unaffected by the lowest iron concentration. The growth rate of 0. tenuis in medium containing the lowest examined level of iron was identical to the rates achieved under iron-satiating growth conditions. Cells grown in media containing 4.1 x and 3.1 x M FeC13had reduced growth rates compared with both the high and low iron grown cells. The recovery of the growth rate of 0. tenuis at the lowest level of iron has been suggested to be linked to the induction of siderophore-mediated iron transport (Brown and Trick 1992). Similar iron-limited growth profiles have been demonstrated in Anacystis nidulans R2, Anabaena variablis, and Synechococcus PCC 7002 (Keny et al. 1988; Wilhelm 1994). In all cases, these cyanobacteria were shown to release extracellular siderophores during periods of iron limitation. In Plectonema boryanum, a filamentous cyanobacteriumthat does not produce siderophores, no such recovery in growth is seen (Keny et al. 1988). For the study of iron-regulated proteins of the outer membrane, we limited our examination to two iron levels, 4.2 x 10-5 M FeC13 for the iron-satiated growth and 5.1 x M FeC13for the low iron growth. A number of polypeptides were induced by growing 0. tenuis at the low iron concentration. The association between the presence of siderophores in the extracellular medium (Brown and Trick 1992) and the membrane proteins that are induced in cells that are grown at low iron levels may be coincidental. By considered siderophore presence, however, some of these proteins may be integral to the iron-transport process. In both Anacystis nidulans (Scanlan et al. 1989) and Synechococcus PCC 7002 (Wilhelm 1994),low iron-inducible peptides have been identified, and at least some of these proteins have been shown to bind to ferrisiderophore complexes (Wilhelm et al. 1996). Periplasmic-binding proteins are often involved in transport functions and typically have high affinities for the substrate

(Ames 1986). It is plausible that the 56-kDa protein released from 0. tenuis on osmotic shock is involved in the periplasmic transport of the ferric ion - catechol siderophore complex. A suggestion that cyanobacteria may employ such a periplasmic transport mechanism is not without precedent. Green and Grossman (1988) and Green et al. (1989) presented evidence that Anacystis nidulans R2 uses a periplasmic transport system to acquire sulfate. The ability of 0. tenuis to grow at low iron levels may be reflected in the complexity of the iron-scavenging and irontransport system: a system composed of at least two extracellular siderophores and the use of iron-regulated outer membrane and periplasmic proteins. Oscillatoria tenuis also appears to respond physiologically to iron limitation; photosynthesis adjusts to iron stress and light-saturated photosynthesis, when normalized to chlorophyll a, is not affected. In this scenario, light-saturated photosynthesis is limited by chlorophyll a content. Similar results have been seen in an activity analysis of photosystems I and I1 (per chlorophyll) in low iron cultured Synechococcus cedorum (Guikema and Sherman 1983). This is in contrast with studies of Aphanocapsa (Sandmann 1985), which showed that in iron-limited cells chlorophyll a either was in excess or associated with nonfunctional or less efficiently functioning photosynthetic units. Iron deficiency does appear to affect photosynthesis more than respiration, potentially as a result of the greater demands on the photosynthetic electron transport chain for iron. This possibility remains to be investigated. Thus, it appears that during periods of iron-limited growth, 0. tenuis is able to assume an alternate physiology that allows the organism to maintain its growth rate with reduced cellular photosynthesis.

Acknowledgements The authors thank Dr. D.P. Maxwell, G.R. Gray, and two anonymous reviewers for comments made in the preparation of the manuscript and the Natural Sciences and Engineering Research Council of Canada for research support.

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