Salt-induced photosystem I cyclic electron transfer restores growth on ...

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[2,3]. More pronounced usage may occur during en- ergetically demanding growth conditions, such as in the case of ... m3P s3I at 34³C in modi¢ed Allen's mineral medium. [4]. ..... [1] Fork, D.C. and Herbert, S.K. (1993) Electron transport and.

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Salt-induced photosystem I cyclic electron transfer restores growth on low inorganic carbon in a type 1 NAD(P)H dehydrogenase de¢cient mutant of Synechocystis PCC6803 Robert Jeanjean a , Sylvie Beèdu a , Michel Havaux b , Hans C.P. Matthijs c , Franc°oise Joset a; * b

a LCB-CNRS, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France Deèpartement de Physiologie Veègeètale et Ecosysteémes, CEA, Centre de Cadarache, 13108 St Paul-lez-Durance, France c ARISE/Microbiologie, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands

Received 29 July 1998 ; accepted 10 August 1998

Abstract The cyanobacterium Synechocystis PCC6803 induces a photosystem I cyclic electron transfer route independent of type 1 NAD(P)H dehydrogenase. The capacity to tolerate raised salinity conditions was shown to operate in a mutant lacking functional type 1 NAD(P)H dehydrogenase. The mutant showed salt-induced enhancement of photosystem I cyclic electron transfer and respiratory capacities. Moreover, this salt-adapted energetic state also restored the capacity of the mutant to grow under inorganic carbon limitation. Uptake of the latter in these conditions became almost as efficient as in the wild-type. The acquired energetic capacities, in contrast, did not allow restoration of photoheterotrophic growth in the type 1 NAD(P)H dehydrogenase mutant. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cyanobacterium; Photosystem I cyclic; Respiration ; NAD(P)H dehydrogenase; Salinity ; Inorganic carbon; Synechocystis PCC6803

1. Introduction Cyclic photosynthetic electron transfer via photosystem I (PSI cyclic) functions in cyanobacteria similarly as in chloroplasts; its operation adds photophosphorylation capacity independently of linear photosynthetic transfer [1,2]. In regular photoautotrophic growth of cyanobacteria, PSI cyclic estab* Corresponding author. Tel.: +33 04 91 16 41 88; Fax: +33 04 91 71 89 14; E-mail: [email protected]

lishes ¢ne tuning of a stable phosphate potential [2,3]. More pronounced usage may occur during energetically demanding growth conditions, such as in the case of cultivation of halotolerant cyanobacteria in high salt medium. In these conditions, signi¢cant increases of PSI cyclic electron £ow and dark respiration have been reported in Synechocystis PCC6803 [4,5] and in other strains [6^8]. A mutant of Synechocystis PCC6803, M55, which lacks the 55-kDa subunit (encoded by gene ndhB) and demonstrates impaired function of the type 1

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 7 2 - 3

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NAD(P)H dehydrogenase (NDH1) complex has been obtained [9]. The main phenotypic trait described for this mutant is inability to grow in the presence of limiting levels of inorganic carbon (Ci) or in photoheterotrophy. The former observation highlights a role for NDH1 in inorganic carbon metabolism, which has been hypothesized to concern energy supply for uptake of the carbon substrate [10]. M55 retains a low level of respiration and cyclic electron £ow capacity [11,12]. The presence of these residual activities supports the potential existence of other routes for PSI cyclic electron £ow that would not proceed via NDH1 [2,13]. In this paper we report that the M55 mutant, in spite of its serious bioenergetic limitations, is nevertheless able to grow in high salt medium. This feature is correlated with capacity changes of the respiratory and PSI cyclic activities. The results suggest that the increased PSI cyclic capacity proceeds via a new pathway, notably in the absence of functional NDH1. The role of NDH1 in adaptation to limiting Ci conditions and in photoheterotrophy will be discussed.

2. Materials and methods 2.1. Strains and culture The wild-type (WT) Synechocystis PCC6803 strain originates from the Pasteur Culture Collection. The NDH1 null mutant M55 (ndhB: :Kmr ) of Synechocystis PCC6803 was kindly provided by T. Ogawa [9]. Only one copy of this gene is present in the genome of Synechocystis PCC6803 [14]. The genotype of M55 was routinely checked by DNA hybridization or PCR. The WT strain and the M55 mutant were cultivated under continuous illumination at a light intensity of approximately 50 Wmol photons m32 s31 at 34³C in modi¢ed Allen's mineral medium [4]. This medium, which contains 50 mM Na‡ ions and 12 mM HCO3 3 , is normal (N) for salt content and high (HC) for inorganic carbon concentration. For high salt conditions (S) the sodium ion concentration was raised to 550 mM by addition of NaCl. For low Ci content (LC) HCO3 3 was omitted from the medium, the only carbon source being CO2 from the air (0.03%) [15]. WT or mutant cells adapted to

normal and high salt regimes will be referred to as WTN or M55N, and WTS or M55S, respectively. Cultures under the HC regime were £ushed with a mixture of 2% CO2 in air. Intermediate Ci concentrations (as indicated in the text) were obtained by addition of bicarbonate to the LC medium. Photoheterotrophic conditions were achieved by addition of 10 WM DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and 1% (w/v) glucose ¢nal concentrations. Adapted cells corresponded to cultivation under the indicated conditions for at least 4^5 generations. Kanamycin was present at 100 Wg ml31 in cultures of M55. Cell density was determined by turbidimetry (one OD580 unit is 1U107 cells ml31 or 4 Wg Chl ml31 ). 2.2. Use of photoacoustic spectroscopy in PSI energy conversion In vivo photochemical energy storage in far-red light ( s 700 nm), indicative of cyclic electron £ow around PSI [1,16,17], was measured with a custommade photoacoustic spectrometer. Cyanobacterial samples (40 Wg Chl) were placed on a membrane ¢lter (Millipore). The samples were illuminated with light passed through a RG715 ¢lter (Schott) to provide PSI light and modulated at 16 Hz. The £uence rate of the resulting far-red light was adjusted using Schott neutral density ¢lters. Photochemistry was saturated with a strong white light of 3000 Wmol photons m32 s31 . Energy storage was calculated as the di¡erence between the maximal (light-saturated) photochemical signal and the actual signal, and was related to the maximal signal as in [17]. Excitation of PSII by the far-red light was insigni¢cant since no e¡ect of DCMU (10 WM) on the energy storage was observed. The photoacoustically monitored energy storage was thus used as a measure of the e¤ciency of the photochemistry at PSI comparable to the quantum yield. 2.3. Redox state of P700 Changes in the redox state of P700, the reaction center of PSI, were monitored in whole cells via absorbance changes around 830 nm, using a modulated £uorescence measurement system MKII (with P700‡ accessory kit) from Hansatech [13]. The cyanobacte-

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rial sample (with 20^40 Wg Chl) was deposited on a membrane ¢lter (Millipore) and placed in the leafclip system of the Hansatech apparatus. The P700 reaction centers were oxidized by illuminating with farred light (730 nm, 8 W m32 ) supplied by a LS2 (high intensity, 100-W tungsten-halogen light source, Hansatech) and an interference ¢lter (730FS10, Corion). For light titrations, samples were illuminated with white light in the presence of 10 WM DCMU; the £uence rate of the white light was then adjusted with a neutral density ¢lter. The kinetics of P700‡ reduction in the dark after interrupting the far-red light or the white light were recorded on a computer. The half-time of P700‡ reduction in the dark was used as a measure of the electron £ow from the cytoplasm towards P700‡ in the cyclic pathway [18]. 2.4. Oxygen exchange O2 evolution was measured in a Clark electrode (Hansatech or Rank Bros.), under various white light intensities provided by a LS2 light source (Hansatech) or by a KL1500 Schott lamp and adjusted by neutral density ¢lters. Cells were previously centrifuged, rinsed twice with LC medium and resuspended in LC medium supplemented with various HCO3 3 concentrations, at a Chl concentration of 10^12 Wg ml31 . O2 consumption in the dark was measured in cell suspensions of 40^60 Wg Chl ml31 . 2.5. Inorganic carbon uptake The rates of Ci uptake were measured by the silicone oil ¢ltration technique [19], using samples of

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5U106 cells in 50-Wl assay mixtures, as in [15]. The cells were pre-incubated in the light until all internal Ci had disappeared. This was estimated by the levelling o¡ of the oxygen evolution rate. During the assay, samples were maintained at 28³C and illuminated under 40 Wmol photons m32 s31 . Incubation times in the presence of 1 mM H14 CO3 3 ranged from 5 to 30 s. 2.6. Miscellaneous Chlorophyll concentration was estimated as in [4]. Solutions of the inhibitors (1000U) were freshly prepared. Flavone (Aldrich) was dissolved in ethanol, and rotenone (Sigma) and DCMU (Cluzeau) in DMSO. Concentrations of inhibitors mentioned in the text are ¢nal ones. In control experiments, the presence of ethanol or DMSO up to 1% (v/v) revealed neither inhibitory nor stimulatory e¡ects (not shown).

3. Results and discussion 3.1. E¡ect of exposure to high salinity on growth rates of M55 cells The growth rate of M55 under N conditions (M55N) (see Section 2.1) was somewhat lower than that of WTN (Table 1). Transfer of WTN or M55N cells, in their exponential phase of growth, from N to S ( = salt added) medium caused a temporary growth arrest of 4^8 h. After this transient period, growth resumed. The growth rates in S medium were lower

Table 1 Generation times, whole cell respiration rates and PSI capacities of WT and mutant M55 cells Type of cells

WTN

WTS

M55N

M55S

Generation times (in h) Dark respirationa +rotenone (20 WM) +£avone (50 WM) Energy storage (in %) Half-time of P700‡ reduction (ms)

5.5 þ 0.5 (3) 390 þ 40 (14) 320 (2) n.d. 16.7 þ 1.9 (14) 300 þ 50 (15)

8.5 þ 0.5 (3) 780 þ 70 (11) 515 (2) n.d. 21.5 þ 3 (12) 180 þ 16 (8)

6.5 þ 0.5 (3) 80 þ 10 (5) 75 (2) 80 (2) 5.9 þ 0.9 (6) 980 þ 60 (5)

10.5 þ 0.5 (3) 175 þ 15 (6) 180 (2) 200 (2) 10 þ 1.7 (4) 580 þ 30 (5)

Data are given for cells adapted to standard (50 mM NaCl, WTN, M55N) or high salt (550 mM NaCl, WTS, M55S) media. Use of rotenone (20 WM), and other methods are presented in Section 2. Energy storage and reduction of P700‡ were determined under 8 W m32 far-red light. Data are mean values of separate experiments (numbers in parentheses) þ S.D. a nmol oxygen mg31 Chl min31 .

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Fig. 1. Growth of WT (F) and M55 (R) cells in HC growth medium with 0.85 M NaCl added.

by about 40% similarly in M55S and WTS (Table 1). Though no growth of M55 was reported at 0.9 M NaCl in BG11 medium [5], its growth could be established in the present medium with NaCl added up to 0.85 M, albeit slightly less e¤ciently as that of WT (Fig. 1).

ment of respiratory activity in M55S can proceed independently of NDH1. In support of the view that another pathway is involved it was demonstrated that the NDH1 inhibitor rotenone (20 WM) provoked an only slightly stronger inhibition of respiration in WTS than in WTN, and furthermore that its addition remained without e¡ect in M55S and in M55N (Table 1). The lack of inhibition of the saltinduced respiratory enhancement by the NDH2 inhibitor £avone (tested in a concentration range of 20^500 WM, encompassing those e¤cient in other organisms [20]) suggested that a standard NDH2 does not mediate the salt-increased activity, even in M55N/S (Table 1). A similar conclusion was recently reached by Tanaka et al. [5] using the same mutant. The actual expression of the potential genes for NDH2 enzymes present in the Synechocystis PCC6803 chromosome [14] and the functional role

3.2. High salt-induced changes of PSI cyclic in strain M55 PSI cyclic activity in M55N cells, measured via di¡erent experimental approaches, corroborated the conclusions from Mi et al. [11,12] that this mutant has limited PSI cyclic capacity (Table 1). However, M55S cells showed an approximate doubling of energy storage and an about two-fold faster reduction of P700‡ . WTS demonstrated a similar increase relative to WTN [4]. These results establish that the electron transfer induced after adaptation to high salinity in WT Synechocystis PCC6803 cells [4] is functional in the absence of NDH1. 3.3. High salt-induced changes of dark respiration in strain M55 Despite the strong reduction of respiratory electron transfer capacity in M55N cells (Table 1 and [11]), adaptation to high salt resulted in a doubling of the dark oxygen uptake rate, very much as in the comparison between WTN and WTS cells (Table 1). As for PSI cyclic electron £ow, this de novo instal-

Fig. 2. Growth of WT and M55 mutant cells in photoautotrophic low Ci (LC) conditions (A) and in photoheterotrophic growth conditions (B). WTN (F), WTS (E), M55N (+) and M55S (R).

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of these proteins in cyanobacterial cell physiology remain to be revealed. 3.4. Restoration of growth of M55 in low inorganic carbon by salt As reported by Ogawa [9] M55N adapted to the standard HC medium were unable to grow in LC conditions. The minimal Ci concentration supporting growth in M55N was established to be 2 mM HCO3 3 . Interestingly, adaptation of M55 cells to high salinity led to a disappearance of the HC phenotype (Fig. 2A). The growth rate of WTS in the LC regime was slightly lower than WTN due to the presence of salt. The growth rate of M55S remained lower than that of WTS cells in the same LC conditions (24 against 14 h, respectively). Very interestingly,

Fig. 3. Oxygen evolution in WT and M55 cells as a function of (A) Ci concentration and (B) light intensity. WTN (F), WTS (E), M55N (+) and M55S (R).

Fig. 4. Ci uptake rates in WTS (F) and M55S (R) cells.

M55S proved to retain the inability of M55N to grow under photoheterotrophic conditions (Fig. 2B). This points to the essential participation of the NDH1-dependent respiratory and/or PSI cyclic pathways in this mode of growth. Lack of re-oxidation of the NAD(P)H co-factors and conceived metabolic constraints could hamper growth. This again suggests that the stimulation of dark respiration capacity taking place in these cells is not proceeding via a salt-induced functional equivalent of type 1 NAD(P)H dehydrogenase. The restoration of LC growth in M55S also became apparent in studies of oxygen production at various Ci supply conditions and with a ¢xed light intensity of 200 Wmol photons m32 s31 (Fig. 3A). M55N showed uptake of oxygen for Ci concentrations below approximately 0.4 mM, whilst above this concentration oxygen evolution remained poor as compared to M55S, WTN and WTS cells. Increases in oxygen evolution with increasing light supply were evident in M55S, WTN and WTS cells (Fig. 3B). In M55N such a light dose-dependent increase was much less evident, as the rate of oxygen evolution strongly lacked behind. From these observations and those presented in Fig. 2A, it could be anticipated that the Ci uptake capacity would become restored in the LC M55S cells. This was indeed observed by measurements of uptake of labelled Ci (Fig. 4). Energy-demanding conditions (high salinity) combined with the absence of NDH1 in Synechocystis PCC6803 have demonstrated the existence of saltinducible PSI cyclic £ow, independent of NDH1.

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This acquired capacity plays a major role not only in supporting growth in high salinity, but also under Ci limitation, and possibly in still other energy-demanding conditions. Complementation of the HC phenotype of M55 by the salt-induced energetic modi¢cations supports the view that PSI cyclic function is essential for the energization of Ci uptake at work under LC conditions [10]. This salt-induced PSI cyclic activity, however, was not su¤cient to restore the growth rate to the level of the WT in similar conditions. Re-oxidation of NAD(P)H co-factors, otherwise e¡ected by NDH1, could be limiting Ci assimilation. However, the salt-induced electron £ow proved inappropriate to support photoheterotrophic growth, for which the genuine NAD(P)H dehydrogenase complex might be essential. Elucidation of the intermediate(s) involved in the operation of this salt-inducible electron £ow is ongoing. Involvement of £avodoxin [21] and FNR [22] can be anticipated, since their synthesis is enhanced in response to occurrence of high salinity in Synechocystis PCC6803.

Acknowledgments

[4]

[5]

[6]

[7]

[8]

[9]

[10]

Mutant M55 was kindly provided by T. Ogawa. The authors wish to thank Ms. A. Janicki for her excellent technical assistance. Gratitude is due to the French/Dutch Van Gogh program which facilitated the cooperation between the Amsterdam and Marseilles laboratories. This work was supported by grants from the CNRS, the Universiteè de la Meèditerraneèe (Ministeére de l'Education Nationale et de la Recherche), the Commissariat aé l'Energie Atomique (CEA), and from the Dutch Society for Life Sciences (SLW), with ¢nancial aid of the Dutch Organisation for Scienti¢c Research (NWO).

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