The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Syn6803) exhibits photomovement through gliding motility. For a better understanding of photomovement in Syn6803, we examined the effects of Ca^sup 2+^ on photoorientation and motility using a computer-assisted videomicroscope motion analysis system. When calcium ion was chelated from the basic motility medium by adding 0.5 mM ethylene glycol-bis-([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the photoorientation was completely inhibited, whereas the gliding motility remained approximately 70% of the control. Photoorientation impaired by EGTA was nearly recovered within 30 min upon addition of 1 mM Ca^sup 2+^. The recovery of photoorientation by Ca^sup 2+^ was mimicked by either Mn^sup 2+^ or Mg^sup 2+^ but not by Ba^sup 2+^ or Sr^sup 2+^. Eanthanum ion at 10 µM completely inhibited both phototactic orientation and gliding motility of Syn6803. Furthermore, pimozide (voltage-gated L-type calcium channel inhibitor), orthovanadate (calcium efflux blocker) and A23187 (calcium ionophore) partially inhibited phototactic orientation and gliding motility. Interestingly, photoorientation was prevented with increasing concentrations of calmodulin antagonist such as trifluoperazine (TFP) and chlorpromazine, but gliding motility was inhibited in proportion to the concentration of TFP. The results we present strongly indicate that Ca^sup 2+^ plays a significant role in regulating the photomovement of Syn6803.
Abbreviations: ATPase, adenosine triphosphatase; CPZ, chlorpromazine; EGTA, ethylene glycol-bis-([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid; Syn6803, Synechocystis sp. PCC 6803; TES, N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid; TFP, trifluoperazine.
The unicellular photosynthelic cyanobactcrium Synechocystis sp. PCC 6803 (Syn6803) exhibits a clear photomovement by light direction or spectral quality (1). In addition, Syn6803 displays a surface-dependent phototactic motility that requires Type-IV pili (2-4). Despite many years of study of the photomovement in Syn6803, the underlying mechanism of how cells sense the direction of light and coordinate their Type-IV pili components appropriately and what environmental factors affect the movement still remains to be solved.
In particular, external cations may control the photomovement in Syn6803. Among the cations, calcium ion has been primarily considered as a messenger or regulator of photosensory transduction. Several lines of experimental evidence indicate that the Ca^sup 2+^ ion significantly influences phototactic responses and motility in eukaryotes (5-7). The effects of cations on phototaxis were first studied in the flagellated algae Platymonas subcordiformis (5). Different concentration ratios of Ca^sup 2+^, Mg^sup 2+^ and K+ in the extracellular medium are able to affect both the phototactic activity and the reversion between positive and negative phototaxis. Further evidence indicated that Ca^sup 2+^ or Mg^sup 2+^ induced optimum motility in Chlamydomonas reinhardtii (6) and in the flagellated alga Euglena gracilis (7), whereas Ca^sup 2+^ and K+ or NH^sub 4^+ were exclusively required for phototaxis (6). In addition, when calcium was depleted from the growth medium, the net phototactic response of a population of cells decreased essentially to zero in C. reinhardtii (8). Other studies have reported that calcium ions are required for phototaxis in Cryptomonas sp. and the slime mold Physarum polycephalum (9,10).
Calcium ions can play a significant role in prokaryotes as they do in eukaryotes, where they regulate motility and other behavioral responses including phototaxis. There are several findings that support the involvement of Ca^sup 2+^ in behavioral responses of prokaryotes (11-13). For instance, calcium ion affects tumbling in bacterial chemotaxis (11,12). An increase in intracellular calcium levels causes Escherichia coll cells to undergo incessant tumbling, thus inhibiting chemotaxis. In Halobacterium halobium, calcium ions are involved in both chemotaxis and phototaxis (13). Submillimolar concentrations of calcium are required for the gliding motility in myxobacteria (14). There are some interesting reports about the photomovement of cyanobacteria (15-17). The involvement of calcium in signal transduction is suggested by the fact that ^sup 45^Ca^sup 2+^ uptake into Phormidium uncinatum is transiently increased after light-to-dark stimulus (17). Such calcium requirement was first demonstrated in the unicellular cyanobacterium Synechococcus strain WH8102, in which the swimming motility requires the outer-membrane glycoprotein SwmA, which contains a number of Ca^sup 2+^ binding motifs (18). In addition, another unicellular cyanobacterium Synechococcus strain WH8113 also exhibits a specific requirement of Ca^sup 2+^ for unusual swimming motility (19).
Possible roles of Ca^sup 2+^ and Ca^sup 2+^-binding proteins in motility were investigated in the extracellular cell walls of P. uncinatum cells. A strain of P. undnatum deficient in an extracellular Ca^sup 2+^-binding glycoprotein called oscillin was unable to move, suggesting that oscillin is responsible for the gliding movement in P. uncinatum (20).
In many cyanobacteria, whether filamentous or unicellular, Ca^sup 2+^ ions are known to affect directional motility, although it is not clear how or where Ca^sup 2+^ acts on the gliding machinery to bring about the changes in motility. In view of this, the role of Ca^sup 2+^ in the photomovement of Syn6803 and the relationship between extracellular Ca^sup 2+^ and intracellular Ca^sup 2+^ signaling are not well understood. Here, we describe Ca^sup 2+^ involvement in phototactic orientation and gliding motility and the potential role of Ca^sup 2+^ as a second messenger in the signaling process of the photomovement of Syn6803. We examined in detail the effects of calcium ion on the photomovement of Syn6803 using a laboratory-developed computer-aided motion analysis system.
MATERIALS AND METHODS
Bacterial strains and culture conditions. The motile strain Syn6803 from Pasteur Culture Collection was cultured as described previously (1). The cells were allowed to glide for 3 days after inoculation on BG11-medium (0.4% agar) at 28°C under lateral illumination with white fluorescence light adjusted to 10 µmol/m^sup 2^/s.[dagger]
Preparation of cell suspension. A small portion of front edges in gliding cell populations were collected separately and then suspended in a hasic motility medium containing 0.24 mM CaCl^sub 2^, 0.3 mM MgCl^sub 2^, 0.46 mM KCl and 18.4 mM NaCl buffered with N-tris[hydroxymethyl]-methyl-2-amino-ethanesulfonic acid (TES) (5 mM, pH 8). Cells were washed once by centrifugation (3000 rpm at 28°C for 3 min) and resuspended in the same medium. In all cases, cell populations thus prepared were incubated in a six-well plate in darkness by gentle shaking for 2 h before transferring to a microscope slide. Immediately after transfer to a new medium, the cells usually showed gliding motility, which persisted for a few hours depending on the different ion composition of the suspension media. Each experiment was repeated three-five times, and the data shown are the average of values obtained together with standard deviations.
Phototaxis and motility assay. Photoorientation and gliding speed of Syn6803 were measured by means of the laboratory-developed computer-aided motion analysis system as described previously in detail (1). A small drop of cell suspension (100 µL) was placed in a quartz chamber (purchased from National Institute for Basic Biology, Okazaki, Japan; 0.5 mm in depth), and a coverslip was pressed over the cell population. The samples thus prepared were allowed to equilibrate to an even population throughout the quartz chamber over a period of about 30 min in darkness and then observed with an upright microscope (Axioskop 50, Carl Zeiss, Eching, Germany) fitted with a 40× objective lens. The microscopic behavior of the cyanobacteria was simultaneously videotaped and stored by image analyzer (IBAS, Carl Zeiss). Data processing and analysis were conducted using the developed analysis software Pathfinder as previously described (1). For the study of cyanobacterial photomovement, individual cell movement was recorded during an initial 5 min in the absence of stimulus light and subsequently for 10 min under stimulus light irradiation. Syn6803 cell suspension was laterally illuminated with red light (660 nm) at a constant fluence rate of 3 µmol/m^sup 2^/s, obtained by means of an Oriel (Stratford, CT) interference filter with a 5 nm half-band width and a lamp source, a 500 W xenon short arc lamp (UXL405S-O, Ushio Electric, Tokyo, Japan). Cell motion parameters (r value for photoorientation and scalar speed for gliding motility) were measured in control and experiment groups.
Treatment of calcium inhibitors. Cells were resuspended in a hasic motility medium containing 0.24 mM CaCl^sub 2^, 0.3 mM MgCl^sub 2^, 0.46 mM KCl, 18.4 mM NaCl and 5 mM TES (pH 8). Ethylene glycol-bis-([beta]-aminoethyl elher)-N,N,N',N'-tetraacetic acid (EGTA) was added at different concentrations (0-5 mM), and the suspension was incubated in darkness for 30 min. Photoorientation and gliding speed were measured for 10 min-path tracks after EGTA-treated cells were stimulated by illumination. For the recovery of photomovement from calcium depletion, cells were exposed to chelator (0.5 mM) at the zero time point, and CaCl^sub 2^ (1 mM) was added 1 h later. Cells were assayed for gliding speed and photoorientation. For each experiment, after washing cells with fresh motility medium, the supernatant was discarded, and the basic motility medium was added to resuspend the pellet to an optical density of 0.4 at 730 nm. In standard assays with Ca^sup 2+^ ionophore (A23187), P-type Ca^sup 2+^-adenosine triphosphatase (ATPase) inhibitor (orthovanadate), Ca^sup 2+^ channel inhibitors (diltiazem, verapamil, pimozide, LaCl^sub 3^) and calmodulin antagonists (trifluoperazine [TFP], chlorpromazine [CPZ]), stock solution of inhibitor was added and incubated for 30 min in darkness before activity was tested. All chemicals and reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The calcium ionophore A23187 was used as a 10 mM stock solution dissolved in ethanol. As a control, an equal amount of dimethyl sulfoxide or ethanol was confirmed to inhibit neither motility nor photoorientation.
Basic motility medium for Syn6803
To investigate the effects of cation(s) on the photomovement of Syn6803, we made a basic motilily medium for Syn6803 (see Materials and Methods). As an initial attempt, we tested whether Syn6803 cells in our basic motility medium show identical phototactic behavior to that of cells in normal growth medium (BG11). Our results showed that Syn6803 cells in the basic motility medium exhibited the same phototaxis (mean r value of 0.83) as that observed in normal medium (mean r value of 0.81) (Table 1). Thus, all the experiments conducted in this study were tested in basic motility medium.
Effects of depletion of specific cations on phototactic response
Subsequently, we examined which of the cations play a crucial role in phototactic orientation and gliding motility.
Because the change of ionic environment can influence the phototactic response, we depleted each ion from the basic motilily medium and studied its effects on photoorientation (r value) and motility (gliding speed). Among specific cations depleted, the removal of calcium inhibited most severely both photoorientation and motility (Table 1).
In the absence of external calcium (calcium-free medium), the photomovement parameters for phototactic orientation and gliding speed were reduced to r values of 0.48 and 4.6 µm/min, corresponding to 58% and 27% of the control, respectively. Microscopic examination of Syn6803 in calcium-free medium indicated that a considerable number of cells tended to stick to the surface of the microscopic slide, resulting in remarkably reduced motility (data not shown). However, the free-moving cells left were still able to move slowly toward the light, showing an r value of 0.48. We also examined the photomovement in the presence of EGTA of varying concentrations. At concentrations above 0.5 mM EGTA the photoorientation of Syn6803 was severely impaired, but the gliding speed decreased to 70% of the control (Table 1, Fig. 1A).
Influences of calcium channel inhibitors and calcium ionophore on phototactic gliding movement
We examined the effects of a number of Ca^sup 2+^ antagonists on the photomovement of Syn6803. The channel inhibitors affected gliding speed as well as phototactic orientation to varying degrees (Table 2). The concentrations of inhibitors other than LaCl^sub 3^ were fixed to 100 µM as used in various motile microorganisms. None of these inhibitors affected cell viability at given concentrations (data not shown).
Verapamil and diltiazem are widely used as voltage-gated calcium channel blockers in eukaryotic cells (21). Whereas verapamil had little effect on the phototactic orientation and the gliding motility of Syn6803, diltiazem reduced only the motility to 77% of the control (Table 2). Another calcium channel blocker, pimozide, decreased phototactic orientation and gliding motility to 47% and 45% of the control, respectively. At 100 µM the calcium ionophore A23187 reduced phototactic orientation and gliding motility to 56% and 49% of the control, respectively. La^sup 3+^, calcium analog and calcium channel blocker inhibited the phototactic response in a concentration-dependent manner (Fig. 1B). We found that lanthanum ion at 10 µM completely inhibited both photoorientation and gliding motility of Syn6803 cells (Table 2, Fig. 1B). The photomovement of Syn6803 was shown to be sensitive to orthovanadate, a phosphate analog known to inhibit the P-type Ca^sup 2+^-ATPase.
Recovery of phototactic orientation by adding divalent cations to Ca^sup 2+^-chelated medium
The specific effects of divalent cations on the recovery of phototactic orientation were plotted up to 4 h after the addition of cation. When calcium chloride (1 mM) was readded to the chelated cell suspension in random motion, the average r value was restored to approximately 0.7 (Fig. 2). The recovery of photoorientation was observed within 30 min of the addition of calcium ion (Fig. 2), and the average gliding speed remained constant for over 15 min after treatment (data not shown). By addition to Ca^sup 2+^-chelated medium of other divalent cations, the recovery of phototactic orientation was observed in the following order: Ca^sup 2+^ > Mn^sup 2+^ > Mg^sup 2+^. However, neither Ba^sup 2+^ nor Sr^sup 2+^ replaced Ca^sup 2+^. The restoration of photoorientation by Ca^sup 2+^ ion indicates that the gliding movement apparatus responsible for phototactic response was reversibly arrested by a short exposure to 0.5 mM EGTA.
Effects of calmodulin antagonists on the photomovement of Syn6803
To evaluate the involvement of Ca^sup 2+^ and calmodulin in the photomovement of Syn6803, cells were preincubated with calmodulin antagonists such as TFP and CPZ. We examined the concentration-dependent effects of phenothiazine derivatives on the photomovement of Syn6803. TFP and CPZ inhibited the phototactic response in Syn6803 cells in a dose-dependent manner (Fig. 3A,B). Both TFP and CPZ have substantial effects on the photomovement of Syn6803 in micromolar concentrations. Interestingly, TFP decreased gliding motility linearly to zero by increasing the concentration to 100 µM (Fig. 3A), whereas CPZ decreased gliding motility to half that of the control (Fig. 3B). At final concentrations of over 20 µM, TFP and CPZ reduced the ;· value to 75% and 83%, respectively (Fig. 3A,B). Thus, TFP can block both phototactic orientation and gliding motility, but CPZ inhibits only phototactic orientation.
The photomovement of Syn6803 is a complex behavior initiated by sensing specific light and actuating a gliding-motor apparatus (i.e. Type-IV pili) to produce changes in motility and phototactic orientation. Because Ca^sup 2+^ is required for cyanobacterial phototaxis (16), it is proposed that specific light may act as a trigger to change the intracellular free calcium either directly or indirectly, which in turn alters motility or phototactic orientation (or both). Therefore, we investigated the phototactic response of Syn6803 as a function of the ionic environment to which the cells are exposed.
Among the cations tested, the phototactic response absolutely depends on the extracellular calcium (Table 1). Both photoorientation and gliding speed were diminished when Syn6803 was incubated in the calcium-deprived medium. In contrast to what was observed in the Ca^sup 2+^-deprived basic motility medium, photoorientation was completely inhibited, but gliding speed remained 70% of the control when EGTA was added to the basic motility medium (Table 1, Fig. 1A). This result is consistent with that of a previous study (9), where the addition of 1 mM EGTA completely inhibited phototactic activity but had only a small effect on the motility in a eukaryote, Cryptomonas. Thus, it seems that the increase of phototactic orientation is possibly caused by the uptake of extracellular Ca^sup 2+^ through a Ca^sup 2+^ influx system (10,22). However, the incomplete inhibition of gliding motility is probably caused by mobilizing Ca^sup 2+^ from a hypothetical Ca^sup 2+^-binding protein or by releasing intracellular Ca^sup 2+^ from the cellular component containing Ca^sup 2+^ (23-25). A recent report on Ca^sup 2+^ release from Syn6803 cells under temperature-stress conditions also suggested the presence of the putative intracellular component containing Ca^sup 2+^ (26). By microscopic observation when Syn6803 cells are incubated in Ca^sup 2+^-free condition, a number of cells appeared to lose their twitching frequency (data not shown). It is a well-known fact that Type-IV pili are necessary for the gliding motility of Syn6803 (2-4). Thus, we suggest that, as seen in the Type-IV pili-dependent pathogenic bacterium Neisseria sp. (27), the adherence of the cyanobacterial pili to the solid surface can be affected by the different microenvironmental situations of intracellular or extracellular calcium ion concentration, but the exact mechanism remains to be elucidated.
Calcium ion has long been proposed to be essential for the phototactic response in prokaryotes (13,16). However, because of the difficulty in directly measuring the intracellular Ca^sup 2+^ levels and the complexity of the photomovement, few reports describing the actual role of intracellular Ca^sup 2+^ on phototaxis have been documented. In the present study, EGTA (Table 1, Fig. 1A) and the Ca^sup 2+^ channel blocker LaCl^sub 3^ (Table 2, Fig. 1B) significantly inhibited the photomovement by changing the homeostatic equilibrium of intracellular Ca^sup 2+^ levels. Interestingly, La^sup 3+^ strongly inhibited both gliding motility and phototactic orientation (Table 2, Fig. 1B). This direct effect may be attributed to the fact that La^sup 3+^ can be taken up to the cell, whereas EGTA remains in the extracellular space as described in plants (28,29). Because LaCL^sub 3^ is also known to inhibit Ca^sup 2+^ fluxes across the plasma membranes, the inhibition of gliding motility and phototactic orientation by La^sup 3+^ suggests that the photomovement of Syn6803 can be mediated by changes in intracellular Ca^sup 2+^ concentration. Therefore, this finding may provide additional evidence for the possible role of intracellular Ca^sup 2+^ as a second messenger in photomovement.
In contrast to LaCl^sub 3^, the poor inhibition by verapamil and diltiazem may be due to the lack of access to the cytoplasmic membrane. Indeed, the lack of inhibition by diltiazem is consistent with that for the motility of Synechococcus sp. WH8113 (19). At a high concentration (100 µM) of a voltage-gated L-type Ca^sup 2+^ channel blocker pimozide, phototactic orientation and gliding motility of Syn6803 were reduced to 47% and 45% of the control, respectively (Table 2). Therefore, another pimozide-sensitive Ca channel distinct from the verapamil-sensitive Ca^sup 2+^ channel is presumably responsible for the light-induced Ca^sup 2+^ influx in Syn6803. However, it remains to be determined whether the photomovement of Syn6803 is mediated by the rise of intracellular Ca^sup 2+^ induced by light.
The calcium ionophore A23I87 had a small effect on the photomovement compared with the effect of La^sup 3+^ (Table 2). Our results are consistent with the weak inhibition of the ionophore A23187 on swimming motility observed in another cyanobacterium, Synechococcus sp. WH8113 (19). In addition, similar evidence for the notable effect of Ca^sup 2+^ ionophore A23187 on the movement has also been demonstrated in the photophobic response of the filamentous cyanobacterium P. uncinatum (30). A23187 drastically impaired the photophobic response but inhibited neither motility nor speed of movement up to a concentration of 20 µM.
Orthovanadate, one of the P-type Ca^sup 2+^-ATPaSe inhibitors, inhibited the phototactic response of Syn6803 by 52% (Table 2). This suggests that a calcium-dependent, vanadate-sensitive Ca^sup 2+^-ATPase exists in Syn6803. Thus, we can predict that there is at least one more vanadate-sensitive Ca^sup 2+^-ATPaSe in Syn6803 as demonstrated in Flavobacterium odoratum (31). A homolog gene encoding P-type Ca^sup 2+^-ATPaSe was cloned from Syn6803, and the protein was overexpressed and characterized in the plasma membrane of E. coli (32,33). Therefore, the calcium channels responsible for calcium influx and ATP-driven calcium efflux system can be considered as a critical gate for determining the photomovement of Syn6803.
We examined whether the calcium ion chelated by EGTA can be replaced with additional calcium or other divalent cation (Fig. 2). Treatment with EGTA impaired phototactic orientation immediately, but the readdition of calcium restored the phototactic orientation. Interestingly, Mn^sup 2+^ or Mg^sup 2+^, but neither Sr^sup 2+^ nor Ba^sup 2+^, could be substituted for CaCl^sub 2^. This result is consistent with our previous result based on an agar assay system in which cation effectiveness for the restoration of photomovement was observed in the following order: Ca^sup 2+^ > Mg^sup 2+^ > Mn^sup 2+^ > Sr^sup 2+^ > Ba^sup 2+^ (34). This is also in accordance with earlier investigations, in which the removal of Ca^sup 2+^ by EGTA abolished the photophobic response and the subsequent addition of Ca^sup 2+^ restored the photophobic response in P. uncinatum (17).
Finally, the observation that the calmodulin antagonists TFP and CPZ inhibited the phototactic response at micromolar concentrations supports the participation of a calcium-binding protein in the photomovement of Syn6803 (Fig. 3A,B). These antagonists inhibit a variety of Ca^sup 2+^-sensitive enzymes at physiologically nonlethal concentrations (10-100 µM) by binding to calmodulin in a reversible Ca^sup 2+^-dependent manner and preventing the interaction of the Ca^sup 2+^-calmodulin complex with its target enzymes (35,36). In contrast to the effect of TFP (Fig. 3A), CPZ had no significant effect on gliding motility at the concentrations that altered photoorientation (Fig. 3B). In the green algae C. reinhardtii, CPZ caused a light intensity-dependent reversal of phototaxis; however, the regulation mechanism of phototaxis in cyanobacteria can be somewhat different from that in the eukaryotic green algae. At low concentrations (3-10 µM), CPZ inhibited phototaxis more effectively than motility (37). In E. coli chemotaxis, CPZ inhibited chemotaxis 10 times more effectively than motility (38). The different actions of these phenothiazine derivatives on phototactic activity can be partly attributed to the different affinities and binding kinetics of TFP and CPZ for calcium-binding protein. In conclusion, intracellular calcium is clearly involved in the regulation of the phototactic motility (by unknown photoreceptor(s), signaling components and type IV-pili) in Syn6803.
Acknowledgements-We thank Dr. Youn-II Park for helpful discussion and Dr. Andrea Todd for careful correction of the manuscript. This research was supported by a grant from the Korea Research Foundation (DP0361) to Youn-II Park, Y.M.P. and J.-S.C.
¶ Posted on the website on 12 November 2003.
[dagger] The fluence rate of white light in fluorescence lamp was calculated by spectral calibration at 500 run with a Yamamya photometer, a gift provided by Dr. Masakatsu Watanabe.
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Yoon-Jung Moon, Young Mok Park, Young-Ho Chung and Jong-Soon Choi*
Proteome Analysis Team, Korea Basic Science Institute, Daejeon, Republic of Korea
Received 14 July 2003; accepted 24 October 2003
* To whom correspondence should he addressed at: Proteome Analysis Team, Korea Basic Science Institute, 52 Yeoeun-dong, Yusung-gu, P.O. Box 41, Daejeon 305-333, Republic of Korea. Fax: 82-42-865-3419; e-mail: email@example.com
Copyright American Society of Photobiology Jan 2004
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