Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313

Isabelle Mary1, Chao-Jung Tu2, Arthur Grossman2 and Daniel Vaulot1

1 Station Biologique, UMR 7127, CNRS et Université Pierre et Marie Curie, BP 74, F-29682 Roscoff cedex, France
2 Carnegie Institution of Washington, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA

Correspondence
Isabelle Mary
mary{at}sb-roscoff.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low- to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.


Abbreviations: HL, high light; LL, low light


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
All living organisms must acclimate to environmental stresses that can potentially damage cellular processes (Asada, 1994). Under extreme environmental conditions many polypeptides will denature (Glover & Lindquist, 1998; Gottesman et al., 1998); they lose their native, functional configuration and tend to aggregate. This process can be reversed to some extent, but when conditions become severe, aggregation can lead to irreversible damage of cellular functions and result in cell death (Herman & D'Ari, 1998). Bacteria respond to adverse environmental conditions by synthesizing a set of ‘stress-associated’ proteins that limit cellular damage by preventing the intracellular accumulation of unfolded and misfolded polypeptides (Lindquist & Craig, 1988). Most stress-associated polypeptides, also called heat-shock proteins (Hsp) because they were first observed in cells exposed to elevated temperatures, are either molecular chaperones or energy-dependent proteases. The chaperones play crucial roles in promoting folding, stabilization, solubilization, renaturation and degradation of polypeptides, and also facilitate the transport of polypeptides into specific cellular compartments and the assembly of multiprotein complexes (Bukau & Horwich, 1998; Gottesman et al., 1997). The energy-dependent proteases perform targeted polypeptide degradation, modulating the availability of regulatory elements and removing non-functional but potentially harmful polypeptides that arise when polypeptides misfold, denature and/or aggregate (Gottesman, 1996). Many of these proteases function in conjunction with chaperones since substrates targeted for degradation may need to be unfolded prior to proteolysis.

Cyanobacteria are an ancient (over 3 billion years old), diverse and ecologically important group of eubacteria that evolved to compete in a wide range of habitats. As the only prokaryotes that perform oxygenic photosynthesis, they are major contributors to biomass accumulation on Earth. Since their apparatus for photosynthesis resembles that of plants, they have been used as model organisms by many scientists to investigate the structure and function of plant-type photosynthesis. Synechocystis sp. PCC 6803 is a freshwater, unicellular, non-nitrogen-fixing cyanobacterium for which the complete nucleotide sequence of the genome has been determined (Kaneko et al., 1996). This naturally transformable strain is one of the most popular organisms for genetic and physiological studies of photosynthesis and environmental gene regulation because of its ability to grow both photoautotrophically and photoheterotrophically. The cyanobacterium Prochlorococcus, the smallest known photosynthetic prokaryote, is a dominant primary producer throughout the oligotrophic oceans (Partensky et al., 1999). It is ubiquitous within the 40°S to 40°N latitudinal band and is found both in surface waters, experiencing light intensities as high as 1500 µmol photons m–2 s–1, and at 150 m depth, where light intensities fall below 1 µmol photons m–2 s–1. The wide distribution of Prochlorococcus in the marine environment probably reflects the existence of several physiologically and genetically distinct ecotypes (Moore et al., 1998). High-light-adapted genotypes occupy the upper, well-illuminated but nutrient-poor part of the water column whereas low-light-adapted genotypes are preferentially found at the bottom of the illuminated layer but in a nutrient-rich environment. These ecotypes differ in their optimal light intensity for growth (Moore et al., 1998), pigment content (Moore et al., 1995), light harvesting efficiency, sensitivity to trace metals (Mann, 2002) and nitrogen utilization ability (Moore et al., 2002). Full genome information is available for Prochlorococcus MED4, which experiences growth in high light (HL) waters of the open oceans, and Prochlorococcus MIT9313, which typically grows in low light (LL) environments and is found deeper down in the water column (Rocap et al., 2003).

Cyanobacteria possess stress-associated genes, encoding chaperones such as DnaK, DnaJ, GroEL or ClpB, and proteases such as ClpP (Glatz et al., 1999). A number of these genes may be induced when cells are shifted to high temperatures (Eriksson et al., 2001) or from LL to HL. In Synechocystis sp. PCC 6803, clpB, htpG, dnaK, groES, groEL, groEL2 and hsp17 transcripts all appeared to accumulate when cells were transferred from 20 to 300 µmol photons m–2 s–1 (Hihara et al., 2001). These results are consistent with experiments performed under similar conditions by Huang et al. (2002). Stress-associated multigene family members appear to be differentially expressed under a variety of physiological conditions (Lindquist & Craig, 1988). As an example, cyanobacteria possess four clpP genes (Porankiewicz et al., 1999; Schelin et al., 2002). clpP1 is monocistronic, while clpP2 and clpP3, similar to the situation in Escherichia coli, appear to be arranged in an operon with clpX (Schelin et al., 2002; Clarke et al., 1998) and clpP4 (also called clpR, encoding a protein that lacks the three active-site amino acids representative of ClpP proteases), respectively (Schelin et al., 2002). Unlike most bacterial Clp proteins, and especially ClpP1 (Clarke et al., 1998), the ClpP2, ClpP3, ClpP4 and ClpX polypeptides did not accumulate to high levels upon cold shock, oxidative stress or HL exposure of Synechococcus sp. PCC 7942 (Schelin et al., 2002). Similarly, while the DnaK2 protein of Synechococcus sp. PCC 7942 exhibited a typical increased accumulation following heat shock, the levels of DnaK1 and DnaK3 did not change following the same treatment (Nimura et al., 2001).

In this study we examined the responses of the groES, groEL, dnaK and clp transcripts in the freshwater cyanobacterium Synechocystis sp. PCC 6803, the marine HL-adapted strain Prochlorococcus MED4 and the marine LL-adapted strain Prochlorococcus MIT9313 following a shift from LL to HL. Using real-time, quantitative RT-PCR, the levels of transcripts were quantified over both the short term (30 min, 1 h) and relatively longer term (3, 6 and 12 h) following the shift to HL, allowing the determination of those heat-shock genes with elevated levels of expression following exposure to HL stress.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culture conditions.
Synechocystis sp. PCC 6803 was cultivated in BG-11 medium (Rippka et al., 1979) buffered with 10 mM Tris/HCl, pH 8·2, at 30 °C. Cultures were bubbled with 3 % CO2 in air and illuminated with 30 µmol photons m–2 s–1 from incandescent bulbs. For HL treatments, cells in the mid-exponential growth phase (OD730~0·8) were diluted with fresh medium to an OD730 of ~0·2. The cells (50 ml cultures) were then placed in a temperature-controlled chamber maintained at 30 °C and exposed to 600 µmol photons m–2 s–1 white light for various lengths of time, as indicated in the text, prior to RNA isolation. Prochlorococcus MED4 and MIT9313 were cultivated in PCR-S11 medium (Partensky et al., 1999) at 20 °C at 6 or 20 µmol photons m–2 s–1. For HL treatments, cells in the mid-exponential growth phase (108 cells ml–1, as measured by flow cytometry) were exposed to 40 or 200 µmol photons m–2 s–1 for various lengths of time prior to RNA isolation. The exact light conditions during the experiments are indicated in Results. Separate experiments were performed to assess the cell response to HL as measured by flow cytometry or gene expression (RT-PCR).

Flow-cytometric analysis.
A 1 ml aliquot of the Prochlorococcus culture was sampled at each RNA sampling time following HL exposure for the analysis of flow-cytometric cell parameters (i.e. cell number, chlorophyll fluorescence) as described by Jacquet et al. (2001). Aliquots were fixed for 10 min with glutaraldehyde (0·25 % final concentration), frozen in liquid nitrogen and stored at –80 °C for later analysis. Samples were analysed with a FACSort flow cytometer (Becton Dickinson) with 488 nm excitation. Cell chlorophyll fluorescence was normalized to that of 0·95 µm fluorescent beads (Polysciences).

RNA isolation.
RNA was isolated from pelleted cells that had been frozen at –80 °C, using a modification of the method of de Saizieu et al. (1998), as described by Bhaya et al. (2000). Briefly, 500 µl acidified phenol and 500 µl NAES (50 mM sodium acetate pH 5·1, 10 mM EDTA, 1 % SDS) were added to pelleted cells from 150 ml cultures; 100 mg glass beads (0·1 µm mean diameter, Bio-Rad) was placed in the suspension, which was then vortexed three times for 20 s each. This was followed by two phenol/chloroform (1 : 1, v/v) and one chloroform extraction. Nucleic acids were precipitated with 2 vols ethanol, resuspended in sterile, double-distilled H2O and then treated for 1 h at room temperature with DNase I (20 U FPLC purified, Amersham), according to the protocol recommended by the manufacturer. DNase-treated samples were extracted with phenol/chloroform (1 : 1), then with chloroform, and the RNA was precipitated from the aqueous phase upon addition of 2 vols ethanol. The final RNA pellet was dissolved in 50 µl 10 mM Tris/HCl pH 8·0, 1 mM EDTA and stored at –80 °C.

Real-time quantitative RT-PCR analysis of gene expression.
For the reverse transcriptase (RT) reaction, 200 ng RNA was incubated with a mixture of PCR reverse primers (4 pmol per 20 µl final vol) (Table 1) for 10 min at 70 °C prior to adding 100 U Superscript II RT (Gibco-BRL). The RT reaction was performed at 42 °C for 1 h and stopped by placing the reaction at 72 °C for 10 min. SYBR Green PCR Master mix (Applied Biosystems) or LightCycler DNA Master SYBR Green I (Roche Applied Science) were used as recommended by the manufacturers; each reaction of 25 µl contained 1 µl of a specific, diluted cDNA (diluted 1/10 relative to the cDNA reaction mixture) and 0·4 µmol l–1 of the appropriate primers.


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Table 1. Primers (5'–3') specific for chaperone, protease and control genes used for quantitative RT-PCR reactions for Synechocystis sp. PCC 6803, Prochlorococcus MED4 and Prochlorococcus MIT9313

 
Sequences of whole genomes of Synechocystis sp. PCC 6803 (GenBank NC_000911), Prochlorococcus MED4 (=CCMP1378) (GenBank BX548174) and Prochlorococcus MIT9313 (GenBank BX548175) were retrieved from public databases.

Transcript levels of the Synechocystis sp. PCC 6803 hsp genes were analysed by real-time RT-PCR using the LightCycler System (Roche Applied Science). The amplification programme consisted of 1 cycle of 95 °C for 60 s followed by 40 cycles of 95 °C for 15 s, 55 °C for 20 s, 72 °C for 40 s. The relative levels of transcripts of hsp genes of the Prochlorococcus strains were analysed by real-time RT-PCR using the ABI Prism 5700 sequence detection system (Applied Biosystems). The amplification programme consisted of 1 cycle of 95 °C with a 60 s hold followed by 40 cycles of 95 °C with a 15 s hold and 60 °C with a 1 min hold. The rps (slr1984) and rnpB transcripts were used as external standards, for Synechocystis and Prochlorococcus respectively, since levels of these transcripts were not altered by HL conditions (Mary & Vaulot, 2003) (Fig. 1).



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Fig. 1. Analysis of groES and control genes by real-time quantitative RT-PCR. Fluorescence vs amplification cycle during HL stress was determined using a 1/10 cDNA dilution. Increases in fluorescence, which were due to the cleavage of the reporter dye as the PCR proceeded, relative to the starting values of delta-normalized reporter fluorescence (Rn), were determined and plotted by the instrument against cycle number. (a) groES and rps (control) for Synechocystis sp. PCC 6803. (b) groES and rnpB (control) for Prochlorococcus MED4.

 
Each quantitative RT-PCR experiment was performed on two distinct biological samples (using separate cultures grown under identical conditions) with three replicates for the first, and two replicates for the second experiment. Quantification of the relative fold change in mRNA levels was calculated using the {Delta}{Delta}CT method normalized against the level of rnpB transcript as described in the Applied Biosystems user bulletin #2 (http://dna-9.int-med.uiowa.edu/RealtimePCRdocs/Compar_Anal_Bulletin2.pdf), using the equation , where CT is the threshold cycle for amplification of hsp or rnpB from LL or HL-stressed cultures.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Synechocystis PCC 6803 was shifted from 30 to 600 µmol photons m–2 s–1 white light as studied before by Bhaya et al. (2000) and used as a reference. The HL-adapted Prochlorococcus MED4 was shifted from 20 µmol photons m–2 s–1 to 200 µmol photons m–2 s–1 conditions and the LL-adapted Prochlorococcus MIT9313 was shifted either from 20 µmol photons m–2 s–1 to 200 µmol photons m–2 s–1 or from 6 µmol photons m–2 s–1 to 40 µmol photons m–2 s–1. The photoacclimation dynamics of Prochlorococcus growth were determined in both the MED4 and MIT9313 strains by monitoring variations in cell number and chlorophyll fluorescence by flow cytometry (Table 2). No significant change in cell number was observed for MED4 following a transfer from LL to HL, in agreement with Jacquet et al. (2001), or for MIT9313 shifted from 6 to 40 µmol photons m–2 s–1. In contrast, a decrease in cell number was observed for MIT9313 shifted from 20 to 200 µmol photons m–2 s–1. For both MED4 shifted from 20 to 200 µmol photons m–2 s–1 and MIT9313 shifted from 6 to 40 µmol photons m–2 s–1, chlorophyll fluorescence did not change for the first 6 h and then decreased after 12 h. In contrast, MIT9313 shifted from 20 to 200 µmol photons m–2 s–1 exhibited a rapid decrease in cell fluorescence 3 h after the shift.


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Table 2. Cell number and chlorophyll fluorescence of Prochlorococcus strains MED4 and MIT9313 as measured by flow cytometry during HL exposure

 
Changes in the level of each hsp mRNA after shifting Prochlorococcus MED4, Prochlorococcus MIT9313 and Synechocystis sp. PCC 6803 from LL to HL were analysed by real-time quantitative RT-PCR. Quantitative RT-PCR data (Tables 3–5) were reproducible, although in some cases, standard deviations were relatively high (0·08>SD/mean{approx}0·37>0·77).


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Table 3. Relative change in hsp transcript levels of Synechocystis PCC 6803 following a shift from LL (30 µmol photons m–2 s–1) to HL (600 µmol photons m–2 s–1), as monitored by real-time quantitative RT-PCR

Values >5 are in bold. Relative values at t=0 are calculated relative to the level of rps.

 

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Table 4. Relative change in hsp transcript levels of Prochlorococcus MED4 following a shift from LL (20 µmol photons m–2 s–1) to HL (200 µmol photons m–2 s–1), as monitored by real-time quantitative RT-PCR

Values >5 are in bold and values >10 are in bold italic. Relative values at t=0 are calculated relative to the level of rnpB.

 

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Table 5. Relative changes in hsp transcript levels for Prochlorococcus MIT9313 as monitored by real-time quantitative RT-PCR following a shift from LL (4 or 20 µmol photons m–2 s–1) to HL (40 or 200 µmol photons m–2 s–1)

Values >5 are in bold and values >10 are in bold italic. Relative values at t=0 are calculated relative to the level of rnpB.

 
Overall, the increase in transcript levels in the Prochlorococcus strains was higher and more sustained over the period examined than that of Synechocystis sp. PCC 6803, although the light shift was more drastic in the latter case. In general, mRNA accumulated later in Prochlorococcus MED4 (6 h in HL) than in Prochlorococcus MIT9313 and Synechocystis sp. PCC 6803 (1 h in HL). In Prochlorococcus MIT9313, the increase in transcript levels was less for the transition from 6 to 40 µmol photons m–2 s–1 than for the transition from 20 to 200 µmol photons m–2 s–1. In the remainder of the manuscript we will only consider the 20 to 200 µmol photons m–2 s–1 transition for the MIT9313 strain, except when explicitly mentioned.

Two genes encoding GroEL have been identified in the genome of the Prochlorococcus strains and Synechocystis sp. PCC 6803. The groEL2 gene is monocistronic and the groEL1 gene is part of a bicistronic operon with groES. In all of these strains, groEL1 and groES showed similar patterns of expression in terms of intensity and timing (Tables 3–5). The strongest response was observed in Prochlorococcus MED4, while both Synechococystis PCC 6803 and Prochlorococcus MIT9313 exhibited moderate increases. Furthermore, the response occurred earlier (after 1 h) in Prochlorococcus MIT9313, in agreement with the cell response data. The groEL2 response was not as pronounced as that of groES/groEL1 and occurred later with respect to the transition time.

The second major chaperone system, the DnaK system, is encoded by multiple dnaK, dnaJ and grpE genes. In contrast to Synechocystis sp. PCC 6803, which has four dnaK genes (sll0086, sll0170, sll1932 and sll0058), the Prochlorococcus strains have only three. The polypeptides encoded by these genes in both Synechocystis sp. PCC 6803 and Prochlorococcus strains are very similar to those of analogous genes identified in Synechococcus sp. PCC 7942 (Nimura et al., 2001). The dnaK2 and dnaK3 genes are well conserved among the three strains. In contrast, the Prochlorococcus dnaK1 displayed low similarities to the analogous gene in Synechocystis sp. PCC 6803. Unlike Synechocystis sp. PCC 6803, which has four dnaJ genes (sll1666, sll1933, sll0093 and slr0897), Prochlorococcus MIT9313 and MED4 possess three and two dnaJ homologues, respectively. dnaJ3 shares strong identity (about 60 %) among the three strains, as does dnaJ2, which has 51 % identity between Synechocystis sp. PCC 6803 and Prochlorococcus MIT9313. In contrast, Prochlorococcus dnaJ1 is not well conserved (about 34 % identity) but may still be the paralogous gene to dnaJ1 of Synechocystis sp. PCC 6803 because it is located close to dnaK3 in both strains. Furthermore, grpE is in the same operon as dnaJ3 in the Prochlorococcus strains, whereas it is in an operon with a dnaK gene (sll0058) in Synechocystis sp. PCC 6803. Among the dnaK transcripts, that of dnaK2 increased strongly in all strains (Tables 3–5). The dnaK1 and dnaK3 transcript levels increased moderately in Prochlorococcus MED4 and Synechocystis sp. PCC 6803. Some of these transcripts, including dnaK2 in Prochlorococcus MED4 and dnaJ3 in Prochlorococcus MIT9313, remained high even after 12 h of HL exposure. The dnaJ3 mRNA level increased dramatically in all strains, whereas dnaJ2 mRNA accumulated to a lesser extent in Prochlorococcus MIT9313. Furthermore, grpE mRNA accumulated only in Prochlorococcus MIT9313 (Tables 3–5).

The clp family of genes in the three strains consists of single clpC and clpX genes and multiple clpB and clpP genes. The ClpP ATP-dependent protease is encoded by four distinct genes. The clpP1 gene appears to be monocistronic while clpP2 and clpP3 are each part of bicistronic operons with clpX and clpP4, respectively. Of the two clpB genes, only clpB1 mRNA levels rose following HL exposure of the three strains. As in Synechocystis sp. PCC 6803, the clpP1 transcript level markedly increased in Prochlorococcus MED4 (>10-fold), whereas clpP2, clpP3 and clpC mRNA accumulated to high levels in Prochlorococcus MED4 only. In contrast, many transcripts encoding Clp protease components, with the exception of clpB1 and clpB2, did not increase following HL exposure of Prochlorococcus MIT9313 (Tables 3 and 4).

Finally, the htpG transcript accumulated in the three strains but to a lesser extent in Prochlorococcus MED4 and Synechocystis PCC 6803 (Tables 3–5).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Light is one of the most important environmental factors that governs cyanobacterial growth. While phenotypic features such as chlorophyll content respond quite slowly to changes in light levels (Table 2), the expression of certain classes of genes may be modulated on a relatively short time scale. This is the case, for example, for the two-component system genes (Mary & Vaulot, 2003) or for the hsp genes examined in the present paper. Cyanobacteria have several hsp-like genes that encode chaperones and specific proteases (Schelin et al., 2002; Glatz et al., 1999). While the number of genes in the compact genome of Prochlorococcus is reduced for some gene categories (e.g. there are only 12 genes encoding two-component systems in MED4, compared to the ~80 genes in this category on the genome of Synechocystis sp. PCC 6803; Mary & Vaulot, 2003), these strains have almost the same number of hsp genes as Synechocystis sp. PCC 6803. The only exceptions are dnaK4, dnaJ4 and the small hsp17 gene (Table 1), which have no equivalent in either of the Prochlorococcus strains, and dnaJ2, which is absent in Prochlorococcus MED4 but present in MIT9313.

The transcripts for dnaJ2 and dnaJ4 do not increase strongly in Synechocystis sp. PCC 6803 during HL exposure, suggesting that they may not play a crucial role in this acclimation process. In contrast, the dnaK4 transcript increased moderately and the hsp17 transcript showed a strong increase in response to HL stress in Synechocystis sp. PCC 6803. Furthermore, the CIRCE (controlling inverted repeat of chaperone expression) binding repressor, HrcA, which is thought to regulate expression of some chaperone genes (Glatz et al., 1997), is absent from the Prochlorococcus MED4 genome but present in the two other strains, which suggests that another mechanism may be involved in the regulation of hsp expression in this HL-adapted strain.

Besides these differences, the three cyanobacterial strains share many features of hsp gene organization and induction. In many cases, the hsp transcripts increase in abundance soon after exposure to HL and then decline during the later stages of acclimation. This probably reflects the capacity of the cells to reach a homeostatic condition in which the physiology of the cell has been modified with respect to the capture and utilization of excitation energy under HL conditions. Such a change is illustrated by the lower chlorophyll fluorescence observed after 12 h of HL exposure in Prochlorococcus (Table 2). Interestingly, when Prochlorococcus MIT9313 cells were shifted from 6 to 40 µmol photons m–2 s–1, there was no significant increase in the level of most hsp transcripts, and little decrease in chlorophyll fluorescence, suggesting that low-amplitude changes in light levels do not induce drastic responses, and that the cells are able to quickly acclimate without major structural rearrangements.

Interestingly, the hsp genes that exhibit the strongest responses to HL exposure [i.e. groEL and groES (Tables 4–6)] also show the most sustained accumulation of transcripts (over the 12 h period following transfer to HL). This is especially apparent with Prochlorococcus MED4 (Table 3). The polypeptides encoded by such transcripts may be very important (either singly or in combinations) for maintenance of cellular function under specific stress conditions, such as HL. Among the three strains examined, groEL1 and groES are arranged in an operon and encode the most-conserved Hsps. In contrast, the GroEL2 polypeptide is much less conserved. The GroEL/GroES system is a major chaperone system in all bacteria and the ways in which this system responds to stress conditions have been extensively studied in cyanobacteria (Hihara et al., 2001; Clarke & Eriksson, 1996; Apte et al., 1998). For example, the transcripts groEL1 and groEL2 have been shown to accumulate in response to elevated temperatures (Kovacs et al., 2001). In all three strains groES and groEL1 showed somewhat similar levels and kinetics of mRNA accumulation, while the level of the groEL2 transcript increased later with respect to the HL exposure (Tables 3–5). This suggests that the two groEL genes may be regulated differently, as hypothesized previously for Synechocystis sp. PCC 6803 (Rajaram et al., 2001).


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Table 6. Comparative induction of hsp transcripts in Synechocystis sp. PCC 6803, Prochlorococcus MED4 and Prochlorococcus MIT9313 following a shift of the cells from LL to HL

+, >5-fold induction; ++, >10-fold induction.

 
While not all dnaK and dnaJ family members are present in the three cyanobacterial strains examined, the common DnaK2 and DnaJ3 polypeptides appear to be highly conserved. Furthermore, HL triggers a pronounced increase in the dnaK2 and dnaJ3 transcripts in all three of the cyanobacteria following HL exposure (see Tables 3–5), suggesting an important role in HL acclimation.

Among the dnaK genes, the dnaK2 transcripts accumulated to a greater extent than those of the other dnaK genes (Tables 3–5). This agrees with previous results showing that, upon shifting Synechococcus sp. PCC 7942 cells to elevated temperatures, the level of DnaK1 and DnaK3 polypeptides remained relatively constant while that of DnaK2 increased (Nimura et al., 2001). Hihara et al. (2001) demonstrated that HL caused increased accumulation of dnaK2 (sll0170) transcripts in Synechocystis sp. PCC 6803. This agrees with the observation that in most eukaryotes, Hsp70s (the DnaK homologues) are represented by a multigene family in which the different family members are differentially expressed (Lindquist & Craig, 1988; Queitsch et al., 2000).

In parallel to what was observed for the dnaK genes, dnaJ3 mRNA accumulated more than that of the other dnaJ transcripts following HL exposure in Prochlorococcus MED4 and MIT9313, and it was the only dnaJ that increased in HL in Synechocystis sp. PCC 6803. Another common feature of the three strains examined in this study is the contiguous genomic locations of dnaJ1 and dnaK3, with the former immediately downstream of the latter. Interestingly, while these transcripts show some increase in HL in the three cyanobacterial strains (with the least change in Synechocystis sp. PCC 6803), these increases are neither marked nor as sustained as observed for dnaK2 and dnaJ3 (Tables 3–5). In Synechococcus sp. PCC 7942, the dnaJ1 homologue was shown to be essential for growth and was detected in thylakoid membranes (Nimura et al., 2001), suggesting that DnaK3 and DnaJ1 might function cooperatively with respect to photosynthesis.

While the ClpPs and ClpC proteases also appear conserved in Synechocystis sp. PCC 6803 and the Prochlorococcus strains, the responses of these genes to HL is much more variable (Tables 3–5). These proteases, one of the best-studied families in E. coli, are composed of regulatory, ATPase/chaperone polypeptides associated with protease subunits. The three cyanobacteria studied here have two different clpB genes, as in Synechococcus sp. PCC 7942 (Eriksson et al., 2001). In the present study, clpB1 transcripts transiently increased while clpB2 mRNA levels did not change following the transfer to HL (Tables 3–5). ClpB1 was shown to be essential for resistance to high- and low-temperature stress in Synechococcus elongatus (Eriksson & Clarke, 1996; Porankiewicz & Clarke, 1997). While ClpB2 has been suggested to be an essential constitutive protein with general chaperone activity (Eriksson et al., 2001), our data indicate that ClpB1 might have a stress-related function. The clpP transcripts also accumulated in Synechocystis sp. PCC 6803 and Prochlorococcus MED4 after HL exposure, especially clpP1. Interestingly, all of the clp transcripts declined in Prochlorococcus MIT9313 following exposure to HL, except for clpP1. ClpP1 was previously shown to be induced strongly during acclimation of Synechococcus sp. PCC 7942 to either low temperature or UV-B, but was not induced by heat shock (Clarke et al., 1998; Porankiewicz et al., 1998). Similarly, neither ClpP2, ClpP3 nor ClpP4 (ClpR) was shown to increase upon heat or cold shock (Schelin et al., 2002; Porankiewicz et al., 1998), whereas all four Clp proteins were induced by HL intensities to different degrees, but again the most pronounced increase was for ClpP1 in Synechococcus sp. PCC 7942 (Clarke et al., 1998). ClpX, ClpP3 and ClpR were found to be primarily constitutive proteins, critical for cell viability in this strain (Schelin et al., 2002). These data suggest the complexity in the control and importance of the different clp genes in cyanobacteria in response to stress conditions. The arrangement of clp genes is conserved in the genomes of several cyanobacteria including Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, Nostoc punctiforme and Prochlorococcus marinus (Schelin et al., 2002). For example, the clpP2 gene is part of an operon with clpX and, therefore, ClpP2 may preferentially associate with ClpX and not with other Clp/Hsp100 proteins, such as ClpC (Porankiewicz et al., 1999). In the present study, clpX mRNA did not accumulate in any of the three strains following HL exposure (Tables 3–5), raising the possibility that ClpX in cyanobacteria may function as an independent chaperone that is not functionally linked to ClpP (Levchenko et al., 1995).

The htpG transcript accumulated in all strains following exposure to HL (Tables 3–5), which is consistent with recent microarray studies (Hihara et al., 2001; Huang et al., 2002). Moreover, it has been shown that HtpG is essential for survival during heat (Tanaka & Nakamoto, 1999) and cold stress (Hossain & Nakamoto, 2002), and is also involved in acclimation of Synechococcus sp. PCC 7942 to oxidative stress (Hossain & Nakamoto, 2003).

Overall, hsp genes were induced to higher levels for longer periods after the light shift for Prochlorococcus MED4 than for either Prochlorococcus MIT9313 or Synechocystis sp. PCC 6803. For example, while in Synechocystis sp. PCC 6803 only the clpP1 transcript level rose substantially following exposure to HL and no clpP transcript accumulated in Prochlorococcus MIT9313, transcripts for all four of the clpPs strongly increased in Prochlorococcus MED4. Furthermore, while transcripts encoding ClpC (Kaneko et al., 1996; Clarke & Eriksson, 1996) declined following HL exposure of Synechocystis sp. PCC 6803 (Eriksson et al., 2001) and Prochlorococcus MIT9313, they showed a sustained increase in Prochlorococcus MED4. Prochlorococcus MED4 is found in ocean surface waters, where light intensities can reach up to 1500 µmol photons m–2 s–1 at midday, nutrient levels can be extremely low (Partensky et al., 1999), and the cells probably experience oxidative stress. Our results for Prochlorococcus MED4 suggest that considerable protein misfolding and aggregation could take place in surface oceanic waters and that hsp genes could be critical for survival in such an extreme environment. Another example of adaptation of Prochlorococcus MED4 to a HL environment is the high number (22) of hli genes encoded by its genome relative to the genomes of the LL-adapted strains (Bhaya et al., 2002); this gene family is critical for cyanobacterial survival during HL exposure (He et al., 2001),

In conclusion, based on expression patterns, at least seven genes, groEL1/ES, groEL2, dnaK2, dnaJ3, clpB1 and clpP1, are suspected to play a key role in the acclimation of cyanobacteria to HL (Table 6). Interestingly, these genes are among the most conserved in the three strains considered. They should be targeted for more detailed analyses including protein expression and genetic studies.


   ACKNOWLEDGEMENTS
 
We wish to thank Florence Le Gall for taking care of the cultures. This study was supported by the European program MARGENES (EU contract QLRT-2001-01226) and by a PhD grant funded by the Région Bretagne. The ABI 5700 instrument was funded by the GenoMer program (Plan Etat Région) and CNRS. This work was also supported in part by an NSF grant INT 990996 awarded to A. R. G.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Apte, S. K., Fernandes, T., Badran, H. & Ballal, A. (1998). Expression and possible role of stress-responsive proteins in Anabaena. J Biosci 23, 399–406.

Asada, K. (1994). Production and action of active oxygen species in photosynthetic tissues. In Causes of Photooxidative Stress and Amelioration of Defence Systems in Plants, pp. 77–104. Edited by C. H. Foyer & P. M. Mullineaux. Boca Raton, FL: CRC Press.

Bhaya, D., Vaulot, D., Amin, P., Takahashi, A. & Grossman, A. (2000). Isolation of regulated genes of the cyanobacterium Synechocystis sp. strain PCC 6803 by differential display. J Bacteriol 182, 5692–5699.[Abstract/Free Full Text]

Bhaya, D., Dufresne, A., Vaulot, D. & Grossman, A. (2002). Analysis of the hli gene family in marine and freshwater cyanobacteria. FEMS Microbiol Lett 215, 209–219.[CrossRef][Medline]

Bukau, B. & Horwich, A. L. (1998). The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366.[Medline]

Clarke, A. K. & Eriksson, M. J. (1996). The cyanobacterium Synechococcus sp. PCC 7942 possesses a close homologue to the chloroplast ClpC protein of higher plants. Plant Mol Biol 31, 721–730.[Medline]

Clarke, A. K., Schelin, J. & Porankiewicz, J. (1998). Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation. Plant Mol Biol 37, 791–801.[CrossRef][Medline]

De Saizieu, A., Certa, U., Warrington, J., Gray, C., Keck, W. & Mous, J. (1998). Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nat Biotechnol 16, 45–48.[CrossRef][Medline]

Eriksson, M. J. & Clarke, A. K. (1996). The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 178, 4839–4846.[Abstract]

Eriksson, M. J., Schelin, J., Miskiewicz, E. & Clarke, A. K. (2001). Novel form of ClpB/HSP100 protein in the cyanobacterium Synechococcus. J Bacteriol 183, 7392–7396.[Abstract/Free Full Text]

Glatz, A., Horvath, I., Varvasovszki, V., Kovacs, E., Torok, Z. & Vigh, L. (1997). Chaperonin genes of the Synechocystis PCC 6803 are differentially regulated under light-dark transition during heat stress. Biochem Biophys Res Commun 239, 291–297.[CrossRef][Medline]

Glatz, A., Vass, I., Los, D. A. & Vigh, L. (1999). The Synechocystis model of stress: from molecular chaperones to membranes. Plant Physiol Biochem 37, 1–12.[CrossRef]

Glover, J. R. & Lindquist, S. (1998). Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82.[Medline]

Gottesman, S. (1996). Proteases and their targets in Escherichia coli. Annu Rev Genet 30, 465–506.[CrossRef][Medline]

Gottesman, S., Wickner, S. & Maurizi, M. R. (1997). Protein quality control: triage by chaperones and proteases. Genes Dev 11, 815–823.[CrossRef][Medline]

Gottesman, S., Roche, E., Zhou, Y. & Sauer, R. T. (1998). The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev 12, 1338–1347.[Abstract/Free Full Text]

He, Q., Dolganov, N., Bjorkman, O. & Grossman, A. R. (2001). The high light-inducible polypeptides in Synechocystis PCC6803. Expression and function in high light. J Biol Chem 276, 306–314.[Abstract/Free Full Text]

Herman, C. & D'Ari, R. (1998). Proteolysis and chaperones: the destruction/reconstruction dilemma. Curr Opin Microbiol 1, 204–209.[CrossRef][Medline]

Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A. & Ikeuchi, M. (2001). DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell 13, 793–806.[Abstract/Free Full Text]

Hossain, M. M. & Nakamoto, H. (2002). HtpG plays a role in cold acclimation in cyanobacteria. Curr Microbiol 44, 291–296.[CrossRef][Medline]

Hossain, M. M. & Nakamoto, H. (2003). Role for the cyanobacterial HtpG in protection from oxidative stress. Curr Microbiol 46, 70–76.[CrossRef][Medline]

Huang, L., McCluskey, M. P., Ni, H. & LaRossa, R. A. (2002). Global gene expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to irradiation with UV-B and white light. J Bacteriol 184, 6845–6858.[Abstract/Free Full Text]

Jacquet, S., Partensky, F., Marie, D., Casotti, R. & Vaulot, D. (2001). Cell cycle regulation by light in Prochlorococcus strains. Appl Environ Microb 67, 782–790.[Abstract/Free Full Text]

Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3, 109–136.[Medline]

Kovacs, E., van der Vies, S. M., Glatz, A., Torok, Z., Varvasovszki, V., Horvath, I. & Vigh, L. (2001). The chaperonins of Synechocystis PCC 6803 differ in heat inducibility and chaperone activity. Biochem Biophys Res Commun 289, 908–915.[CrossRef][Medline]

Levchenko, I., Luo, L. & Baker, T. (1995). Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev 9, 2399–2408.[Abstract]

Lindquist, S. & Craig, E. A. (1988). The heat-shock proteins. Annu Rev Genet 22, 631–677.[CrossRef][Medline]

Mann, N. H. (2002). Phages of the marine cyanobacterial picophytoplankton. FEMS Microbiol Lett 27, 17–34.

Mary, I. & Vaulot, D. (2003). Two-component systems in Prochlorococcus MED4: genomic analysis and differential expression under stress. FEMS Microbiol Lett 226, 135–144.[CrossRef][Medline]

Moore, L. R., Goericke, R. & Chisholm, S. W. (1995). Comparative physiology of Synechococcus and Prochlorococcus: influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Mar Ecol Prog Ser 116, 259–275.

Moore, L. R., Rocap, G. & Chisholm, S. W. (1998). Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393, 464–467.[CrossRef][Medline]

Moore, L. R., Post, A. F., Rocap, G. & Chisholm, S. W. (2002). Utilization of different nitrogen sources by the marine cyanobacteria Prochlorococcus and Synechococcus. Limnol Oceanogr 47, 989–96.

Nimura, K., Takahashi, H. & Yoshikawa, H. (2001). Characterization of the dnaK multigene family in the cyanobacterium Synechococcus sp. strain PCC7942. J Bacteriol 183, 1320–1328.[Abstract/Free Full Text]

Partensky, F., Hess, W. R. & Vaulot, D. (1999). Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol Mol Biol Rev 63, 106–127.[Abstract/Free Full Text]

Porankiewicz, J. & Clarke, A. K. (1997). Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 179, 5111–5117.[Abstract]

Porankiewicz, J., Schelin, J. & Clarke, A. K. (1998). The ATP-dependent Clp protease is essential for acclimation to UV-B and low temperature in the cyanobacterium Synechococcus. Mol Microbiol 29, 275–283.[CrossRef][Medline]

Porankiewicz, J., Wang, J. & Clarke, A. (1999). New insights into the ATP-dependent Clp protease: Escherichia coli and beyond. Mol Microbiol 32, 449–458.[CrossRef][Medline]

Queitsch, C., Hong, S. W., Vierling, E. & Lindquist, S. (2000). Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12, 479–492.[Abstract/Free Full Text]

Rajaram, H., Ballal, A. D., Apte, S. K., Wiegert, T. & Schumann, W. (2001). Cloning and characterization of the major groESL operon from a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31. Biochim Biophys Acta 1519, 143–146.[Medline]

Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 1–61.

Rocap, G., Larimer, F. W., Lamerdin, J. & 21 other authors (2003). Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424, 1001–1002.[CrossRef][Medline]

Schelin, J., Lindmark, F. & Clarke, A. K. (2002). The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255–2265.[Abstract/Free Full Text]

Tanaka, N. & Nakamoto, H. (1999). HtpG is essential for the thermal stress management in cyanobacteria. FEBS Lett 458, 117–123.[CrossRef][Medline]

Received 8 January 2004; accepted 13 January 2004.