Analysis of cyanobacterial and plastid deduced Ycf26 protein sequences

The deduced amino acid sequences for Ycf26 from 15 available cyanobacterial annotated genome sequences and the red algal species Porphyra purpurea, Gracilaria tenuistipitata and Cyanidium caldarium were analysed by Pfam and SMART. This identified a HAMP, PAS (low confidence in Cyanidium), HisKA and HATPase_c domain (Figure 1). SMART and Interpro also identified two potential transmembrane helical regions upstream of the HAMP domain (33–55 and 201–223 in 6803). The identification of transmembrane sequences by SMART is with an accuracy of 97–98% [23]. Because the 145 amino acid region that is flanked by the two transmembrane regions is highly conserved in all 15 cyanobacterial sequences and Porphyra (see additional figure online and Prodom Family PD339187 [24]) and it is adjacent and upstream of a HAMP domain (which is known to in most cases be actively involved in transmission of signals from a periplasmic signalling domain to the cytoplasmic portion of the protein [12, 25, 26]), we think it probably functions as a periplasmic sensor. We have not been able to detect homologous sequences in any other protein sequence, suggesting that it is unique to Ycf26 (DspA/NblS). The PAS sensor domain may bind a redox sensitive cofactor [13] that could potentially report on the metabolic state of the cell. Phylogenetic analysis (Ashby, MK and Houmard, J unpublished) and ClustalW alignments of these sequences and the alignment scores indicate that all these proteins (except Cyanidium caldarium and Gracilaria tenuistipitata) are probably orthologues (see supplementary data online). This is supported by Tu et al., [19] who were able to rescue their dspA null mutant in Synechocystis 6803 with the nblS gene from Synechococcus 7942.

Phenotype of the pta, dspA and pta/dspA mutants under nutrient-replete conditions

Mutants were routinely characterised by whole cell absorption spectra, which can be used to give an estimate of the amount of chlorophyll and phycocyanin per cell [27]. Mutants were further characterised by 77 K fluorescence emission spectra. These spectra give an indication of changes in the PSII:PSI ratio [28].

When cells were grown under low light (white light at 10 μmol m^-2 s^-1) the absorption spectra were similar, indicating little difference in the chlorophyll and phycocyanin content of the cells (data not shown). When cells were grown under higher light (80 μmol m^-2 s^-1), only slight differences in absorption spectra and Chl/PC ratios were seen (Table 2). However low-temperature fluorescence spectra recorded with chlorophyll excitation revealed changes in the ratio of PSII/PSI (Figure 2). These spectra show peaks at 685 nm and 695 nm (both from PSII) and 725 nm (PSI) [28, 29]. Spectra have been normalised to the PSI fluorescence emission, as their absolute amplitudes are unreliable [28]. When cells were grown at 80 μmol m^-2 s^-1, the dspA and pta single mutants show a lower PSII/PSI ratio than the wild-type, and this difference was more pronounced in the pta/dspA double mutant (Figure 2). A similar pattern was seen in cells grown at 10 μmol m^-2 s^-1, but the differences were less pronounced (not shown).

Strain	Chlorophyll/Cell (molecules × 106)		Phycocyanin/Cell (molecules × 106)		Chlorophyll/phycocyanin
	+nitrate	-nitrate	+nitrate	-nitrate	+nitrate	-nitrate
Wild-type	13.6 ± 2.1	*	5.6 ± 1.4	*	2.4 ± 0.1	*
pta	12.6 ± 0.4	*	4.6 ± 1.5	*	2.7 ± 0.1	*
dspA	13.8 ± 0.1	*	5.9 ± 0.5	*	2.4 ± 0.1	*
pta/dspA	15.8 ± 0.9	7.8 ± 0.5	5.9 ± 0.5	1.0 ± 0.2	2.7 ± 0.2	7.8 ± 2.3

Phenotype of the pta, dspA and pta/dspA mutants after nitrogen starvation

An nblS mutant of Synechococcus 7942 shows a pronounced phenotype under nitrogen starvation. In the wild-type, cells gradually lose their photosynthetic pigments under these conditions. However, an nblS mutant is bleached to a much lesser extent, retaining its phycobilisomes in particular [21]. We therefore looked at the pigmentation of our Synechocystis 6803 mutants during growth in modified BG11 medium supplemented with glucose (to help maintain the viability of the cells), but containing no NaNO₃.

After three days nitrogen starvation in moderate light (80 μmol m^-2 s^-1), the wild-type and the dspA and pta single mutants were almost completely bleached (Table 2). We could detect no significant differences (less than 5%) in the rate of bleaching during nitrogen starvation in the three strains (data not shown). By contrast, the pta/dspA double mutant retained significant levels of pigment (Table 2). Both the chlorophyll and phycocyanin per cell dropped somewhat compared to the start of the treatment, but loss of phycocyanin was much greater (Table 2). Thus only the pta/dspA double mutant shows a 'non-bleaching like' phenotype: Chlorophyll concentration decreases by a factor of about two and that of phycocyanin by about five. Figure 3 shows low-temperature fluorescence emission spectra for the pta/dspA double mutant grown after 3 days nitrogen starvation. Whether with or without nitrate, it exhibits a similar spectrum meaning that the starved cells retain both PSI and PSII. The starved pta^-/dspA^- cells also retained significant levels of oxygen evolution (data not shown). Thus the pta/dspA double mutant retains its photosynthetic apparatus during nitrogen starvation, in contrast to the wild-type and both single mutants, where nearly all pigment is lost.

A potential new sensor domain in DspA/Ycf26

Ycf26 may have a periplasmic sensor domain, which could play an important role in sensing stress either directly or indirectly (Figure 1). The putative periplasmic sensor and PAS domain may combine to enable Ycf26 to sense a wide variety of stress conditions [[15, 17–19, 21, 22] & this work]. It remains to be determined whether DspA senses any of these signals directly or via other proteins or phosphorelays and the precise way in which these sensor domains interact. This apparent central role of Ycf26 in the regulation of gene expression in response to a number of different stresses is supported by its universal occurrence in the 15 available cyanobacterial genome sequences, particularly the 3 Prochlorococcus species which only have 4–6 histidine kinases in their genome ([30], MK Ashby & J Houmard, unpublished).

Roles of DspA and acetyl phosphate in cell signalling

We have constructed Synechocystis 6803 mutants deficient in the pta gene coding for phosphotransacetylase and the dspA gene coding for the DspA sensory histidine kinase (otherwise known as Ycf26 or NblS), and a double mutant deficient in both genes. We have used the mutants to examine the possible roles of DspA and acetyl phosphate in controlling cell responses to nitrogen starvation. We find strong evidence that DspA and acetyl phosphate act cooperatively as signalling inputs into pathways controlling photosystem stoichiometry under nutrient-replete conditions, and photosystem degradation during nitrogen starvation. Under nutrient-replete conditions, the dspA and pta single mutants both show a decreased PSII/PSI ratio (Figure 2). The PSII/PSI ratio becomes even lower in the pta/dspA double mutant, indicating additive effects of the two mutations. Since acetyl phosphate is known to act as a phosphodonor to response regulators [11], we propose that PSII/PSI ratio is influenced by gene expression controlled by one or more response regulators (or hybrid kinases) whose phosphorylation level is controlled by phosphotransfer both from DspA and from acetyl phosphate. This scheme (in its simplest form) is illustrated in Figure 4.

The effects seen under nitrogen starvation are even more striking. After 3 days' nitrogen starvation under moderate light, wild-type cells are virtually devoid of pigment, indicating almost complete loss of the phycobilisomes and the chlorophyll-protein complexes (Table 2). In this case, the dspA and pta single mutants both behaved in the same way as the wild-type, but the pta/dspA double mutant retained a significant proportion of its photosynthetic complexes, in particular the chlorophyll-protein complexes. Therefore the pta/dspA double mutant is clearly deficient in a signalling pathway required to initiate degradation of these complexes under these conditions. Again, we suggest that this pathway involves gene expression controlled by one or more response regulators whose phosphorylation level is determined by phosphotransfer from both DspA and acetyl phosphate (Figure 4). In this case the double mutation is needed to produce a detectable phenotypic effect, suggesting that either DspA or acetyl phosphate alone are sufficient to maintain high enough levels of phosphorylation of the relevant response regulator(s) (Figure 4). The fact that the phenotypic difference is seen only in the double mutant very strongly suggests that DspA and acetyl phosphate are acting as phosphodonors to the same response regulator(s). Again, it appears that a high level of response regulator phosphorylation promotes the degradation of the photosynthetic complexes.

Acetyl phosphate as a potential global regulator of gene expression in Synechocystis 6803

In E. coli the concentration of acetyl phosphate has been shown to be dependent on the metabolic state of the cell as well as the growth phase, carbon source, pH and temperature [8, 31, 32], therefore acetyl phosphate could potentially serve as a global sensor of the physiological state of the cell in E. coli. Acetyl phosphate is known to phosphorylate several RR's, including CheY, NR_I, PhoB and OmpR [8]in vivo, but its ability to phosphorylate response regulators in vivo depends on the K _m of the response regulator for acetyl phosphate, which in many cases is too high to allow phosphorylation [33]. In the four systems studied in one investigation of crosstalk between two component systems, one, the Uhp system was shown to be activated by acetyl phosphate when E. coli is grown on pyruvate [34]. There have been no comparable studies in cyanobacteria, but it seems likely that the level of acetyl phosphate would change in response to physiological stress, in particular nutrient deprivation. If this is the case, it seems likely that the K _m of at least some response regulators could be tuned to the physiological concentrations of acetyl phosphate in Synechocystis 6803, allowing the level of acetyl phosphate to be part of the signalling process. The level of pigment complexes in the pta mutant was close to normal in nutrient replete conditions (table 2), indicating that the level of acetyl phosphate is not having an effect on overall expression of photosynthetic gene expression via a number of response regulators, but we cannot rule out that the cumulative effect of pta^- and dspA^- that we observe in nutrient depleted conditions may be influenced by a reduced phosphorylation of response regulator(s) other than those phosphorylated by DspA.

Role of DspA in initiating degradation of photosynthetic complexes during nutrient stress in Synechocystis 6803

NblS (DspA homologue) was originally identified in Synechococcus 7942 as a factor important for initiating the degradation of the photosynthetic complexes (in particular the phycobilisomes) during nitrogen starvation [21]. In Synechocystis 6803 there is strong evidence for DspA as a global regulator controlling the expression of many genes involved in photosynthesis, carbon metabolism and stress responses [15, 17–19, 21, 22]. However, dspA mutations in Synechocystis 6803 have not previously been shown to induce a non-bleaching phenotype like that seen in Synechococcus 7942. Here we have shown that such a phenotype can be seen in the dspA mutant of Synechocystis 6803, provided that the pta gene is also deleted. The phenotype is still not identical to that seen in Synechococcus 7942, however, since in Synechocystis 6803 it is not only the degradation of the phycobilisomes which is affected but also the degradation of chlorophyll-protein complexes (Table 2).

Strain and culture conditions

Sequence analysis

Deduced amino acid sequences were obtained form Cyanobase [37], the National Centre for Biotechnology Information (NCBI) [38] and the DOE Joint Genome Institute [39]. Protein to protein BLASTP 2.2.6/3 searches were performed at NCBI and Cyanobase websites. Protein sequences were analysed by Pfam [40, 41], SMART [23, 42] and Interpro [43]. CLUSTALW alignments [44] were performed at the European Bioinformatics Institute [45].

Molecular biology

Routine DNA manipulations were performed as in [46] using reagents purchased from QIAGEN, New England Biolabs, Inc., Promega and Sigma. Genomic DNA from Synechocystis 6803 was prepared as described by Porter [47]. The primers used for PCR amplification of the dspA gene had the sequences 5'-gcgagctcttctgtgtccaatccaacg and 5'-gcggtaccatggattgatacacggccag. The same primers were used to monitor segregation in the transformant. The primers used for PCR amplification of the pta gene had the sequences 5'-GCGAGCTCCCTTTATTTAAGCACCACC and 5'-GCGGTACCTTGCAAAGCTGTAATTACCACC and were also used to monitor segregation in the transformants. Primers were purchased from Invitrogen Technologies.

Construction of dspA and pta mutants of Synechocystis 6803

The pta open reading frame (slr2132 in the Cyanobase database) and the dspA open reading frame (sll0698) were amplified by PCR from Synechocystis 6803 DNA, using primers shown above. The PCR products were cloned into pBluescriptII KS +. The slr2132 sequence was cut at a Pst I site at position 872 in the coding sequence, and an erythromycin resistance cassette (pBSEmEco1) was ligated in. The sll0698 sequence was cut at an Eco RI site at position 1384 in the coding sequence, and a kanamycin resistance cassette (pUC4K) was ligated in [48, 49]. Slr2132 is 265 bp downstream of slr1888 and it is probably transcribed on an mRNA encoding only Pta, so its interruption should not have any polar effects. Sll0698 is upstream of two putative transposases sll0699 and sll0700. The glucose-tolerant strain of Synechocystis 6803 was transformed with the slr2132::Em^R construct as described by Porter [47]. After several rounds of growth on erythromycin plates, PCR was used to check the insertion. This confirmed that the wild-type slr2132 locus had been completely replaced by the slr2132::Em^R locus, and hence that the pta gene had been completely inactivated generating a fully-segregated pta mutant. A similar procedure was followed with the sll0698::Km^R construct, which was used to transform both the wild-type and the pta mutant, to generate a sll0698 single insertional mutant (dspA) and a sll0698:slr2132 double insertional mutant (pta/dspA). These mutants were shown to have segregated after six rounds of streaking to single colonies on kanamycin (plus erythromycin for pta double mutants) plates as has been observed by Hsiao et al. [22], though without glucose in this work.

Procedure for initiating nutrient deprivation

50 ml cultures were grown to mid-log phase in BG11 medium. Each culture was then harvested by centrifugation, resuspended in 30 ml of wash medium, harvested a second time and resuspended in 10 ml of BG11 medium supplemented with glucose but lacking NaNO₃. This was then divided into two 5 ml portions, which were then used to inoculate parallel 20 ml cultures.

Pigment content analysis

Fluorescence emission spectra were measured at 77 K in a Perkin-Elmer LS50 luminescence spectrometer equipped with a liquid-nitrogen sample holder. Cells were harvested by centrifugation and resuspended in growth medium to a chlorophyll a concentration of 5 μM [28]. Re-absorption of emitted fluorescence was negligible at the cell concentrations used. The samples were not mixed with glycerol, as this alters the low temperature fluorescence emission spectrum [29]. The samples were then injected into silica tubes (2 mm internal diameter) and dark adapted for 5 minutes. Samples were rapidly frozen by immersion in liquid nitrogen. Excitation and emission slit widths were 5 nm. Chlorophyll a was excited at 435 nm: light at this wavelength is not significantly absorbed by the phycobilisomes [50]. Chlorophyll a concentration was estimated from the absorption of methanol extracts at 665 nm [51]. Spectra from different samples were normalised to the PSI peak (emission at 725 nm). Cell absorption spectra were recorded with an Aminco DW2000 spectrophotometer. The concentrations of chlorophyll and phycocyanin were estimated from the spectra using the equations of Myers et al. [27]. Cell numbers were estimated from the optical density at 750 nm, calibrated using a hemocytometer.