Abundance Changes of the Response Regulator RcaC Require Specific Aspartate and Histidine Residues and Are Necessary for Normal Light Color Responsiveness

Strains, growth conditions, and transformation.A short-filament F. diplosiphon mutant designated SF33 (12) with a normal CCA response was used as the wild type. Cells were cultured and were spectrally analyzed using a Beckman DU640B spectrophotometer as previously described (36). Cells were grown using light intensities of approximately 15 μmol photons m⁻² s⁻¹ unless indicated otherwise and were transformed by electroporation as described previously (29).

Plasmid construction.RcaE H430 was changed to alanine (A) using primers 5′ RcaEmutHbox (Table 1) and 3′RcaEmutHbox. Primers 5′ RcaE and 3′RcaEmutHbox were used to amplify the 5′ region of rcaE encoding a protein with the H at position 430 replaced by A, while primers 3′ RcaE and 5′RcaEmutHbox were used to amplify the 3′ region of rcaE with the same H-to-A mutation. The two PCR products were combined and used as a template to amplify the whole rcaE coding sequence along with 468 bp upstream of the start codon and 132 bp downstream of the stop codon using the 5′ RcaE and 3′ RcaE primers. The PCR product was cut with BamHI and inserted into pPL2.7 (11). A clone with rcaE transcribed in the same direction as the kanamycin resistance gene was selected, designated pPLrcaEA430, and used to transform the rcaE null mutant FdBk14 (26, 39) to create mutant RcaEH430A.

Plasmids pPLrcaC and pPLN51/Q316 (29) were used as templates to PCR amplify either the wild-type rcaC coding sequence or the same coding sequence with D51 and H316Q mutations plus 768 bp of the upstream promoter sequence. The primers used in the PCRs were rcaC 3XFLAG 5 PstI and rcaC 3XFLAG 3 XmaI. The W03 plasmid contained a 3XFLAG tag sequence followed by a stop codon after the XmaI site. W03 was cut with PstI and XmaI, and the 4.7-kbp fragment containing the 3XFLAG tag was gel purified and ligated with the PCR products described above cut with the same enzymes to create W03rcaC 3XFLAG and W03N51/Q316 3XFLAG. In these plasmids, the 3XFLAG tag (N terminus DYKDHDGDYKDHDIDYKDDDDK C terminus) was fused in frame to the C terminus of the rcaC coding sequence with a proline and a glycine in between. Each of these plasmids was then used as a template with the same upstream primer, rcaC 3XFLAG 5 PstI, and another downstream primer, rcaC 3XFLAG 3 PstI, to PCR amplify the rcaC coding sequence, including the promoter region and the 3XFLAG tag sequence. The PCR products were cut with PstI and inserted into PstI-cut pPL2.7 to make the autonomously replicating plasmids pPL3XFrcaC and pPL3XFN51/Q316. Clones with rcaC transcribed in the same direction as the kanamycin resistance gene were used for transforming wild-type cells and further analysis.

Primers rcaC copper 5 and rcaC copper 3, as well as plasmids pPLrcaC and pPLN51/Q316/N576 (29), were used to PCR amplify the rcaC coding sequence (either the wild-type sequence or a sequence containing the D51N, H316Q, and D576N mutations). The PCR products were cleaved with NdeI and SacI and inserted into the same sites of pGEM-T-PpetE (8) to generate pGEMTrcaC and pGEMTN51/Q316/N576. Primers PstIPpetEUp and PstIPpetELO were used to PCR amplify the petE promoter followed by either the wild-type or mutated rcaC coding sequence, and the PCR products were inserted into PstI-cut pPL2.7 to make pPLPpetErcaC and pPLPpetEN51/Q316/N576. Clones with rcaC transcribed in the direction opposite the direction of the kanamycin resistance gene were selected for transformation into a rcaC null mutant (CR2) and analyzed further.

The DNA that was derived from PCR amplification of all of the constructs described above was sequenced to ensure that no mutations were present in the amplified sequences or at the ligation junctions. All plasmids and strains used in this study are shown in Table 2.

Immunoblot analyses.Extraction of total cellular protein and Western analyses with anti-RcaC antiserum were performed as previously described (29). Western analyses using the anti-FLAG M2-horseradish peroxidase antibody (Sigma, St Louis, MO) were performed according to the manufacturer's instructions, with the following modifications. Membranes were blocked with 5% nonfat milk in TBS buffer (20 mM Tris-HCl [pH 7.5], 140 mM NaCl) containing 0.05% Tween 20 at room temperature for 1 h and then incubated with anti-FLAG M2-horseradish peroxidase antibody diluted 1:1,000 with TBS buffer containing 0.05% Tween 20 overnight at 4°C. The membranes were then washed six times for 5 min each time with TBS buffer containing 0.05% Tween 20. To detect the antibody, SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) was used according to the manufacturer's instructions, and the images were viewed and quantified using a Kodak Image Station 440.

Kinetics of RcaC abundance change.Wild-type cells were grown in RL or GL to an optical density at 750 nm (OD₇₅₀) of approximately 0.7. Cultures were then switched from RL to GL or from GL to RL. Parallel cultures left in either RL or GL were used as controls. At 0, 4, 10, 16, 22, and 28 h after the light switch, three independent replicates of 50-ml cultures of cells exposed to either constant RL or constant GL or subjected to light color switches were harvested by centrifugation at 5,000 × g for 5 min at 4°C. The cell pellets were resuspended in 3 ml of buffer B (50 mM Tris-HCl [pH 7.5], 10% [wt/vol] glycerol, 0.3 M NaCl, 0.05% [wt/vol] Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine, 50 μM 6-amino-n-hexanoic acid) and then lysed by two passages through a French pressure cell at 18,000 lb/in² at 4°C. The cell lysates were then centrifuged at 50,000 × g for 30 min at 4°C. For electrophoretic analyses, 100 μg of total protein per lane was loaded onto 12.5% sodium dodecyl sulfate-polyacrylamide gels. Two duplicate gels were loaded and electrophoresed. After electrophoresis, one gel was stained with Coomassie brilliant blue R-250, and the other was transferred to a nitrocellulose membrane and used for Western blot analysis using anti-RcaC antiserum.

Kinetics of phycobiliprotein abundance change.Wild-type cells were grown in RL or GL to an OD₇₅₀ of approximately 0.6 and then either switched from RL to GL, switched from GL to RL, or left in the original light. Phycobiliprotein and chlorophyll contents were determined essentially as previously described (4, 24, 38). For each treatment, six 1.5-ml samples of cells were collected at each time point from cells exposed to the four different light conditions and centrifuged at 13,000 × g for 1 min at room temperature. The pellets were then frozen in liquid nitrogen. For each replicate, three of the pellets were used to determine the chlorophyll concentration by adding 500 ml of 100% methanol, mixing the preparation, and incubating it in the dark for 1 h at 4°C, which was followed by centrifugation at 10,000 × g for 10 min. The extraction procedure was repeated once, the supernatants were pooled, and the absorbance at 665 nm was recorded. The remaining three pellets were each resuspended in 1 ml of STES (50 mM Tris-HCI [pH 8.0], 50 mM NaCl, 10 mM EDTA [pH 8.0], 250 mM sucrose) supplemented with 5 mg/ml lysozyme. Samples were incubated at room temperature for 30 min, and after centrifugation at 13,000 × g for 5 min, the supernatants were collected. The supernatant absorbance at 565 nm, 620 nm, and 650 nm were used to calculate PE, PC, and allophycocyanin (AP) concentrations as previously described (38). After the averages for three sample values were normalized on the basis of the chlorophyll concentrations, the PE/AP and PC/AP ratios were calculated.

Copper induction. rcaC null mutant (CR2) (29) cells transformed with pPLPpetErcaC or pPLPpetEN51/Q316/N576 were grown to an OD₇₅₀ of 0.7 in BG-11 containing 10 μg/ml kanamycin in the presence of 50 μmol photons m⁻² s⁻¹ GL. Cells were harvested by centrifugation at 5,000 × g for 5 min at room temperature, and then they were resuspended in fresh BG-11 that contained 10 μg/ml kanamycin and either lacked copper sulfate or contained various concentrations (see below) of copper sulfate and were grown in plastic culture tubes in RL and GL. Cells were harvested at an OD₇₅₀ of 0.8, and the total cellular protein was extracted and analyzed as described previously (29).

qRT-PCR amplification analyses.RNA was isolated from wild-type cells grown in RL and GL as described previously (36). The concentration and purity of the RNA were determined spectrophotometrically using a Beckman DU640B spectrophotometer. Purified RNA was treated with a Turbo DNA-free kit (Ambion, Austin, TX) to remove genomic DNA according to the manufacturer's instructions. Reverse transcription was performed by incubating 5 μg of RNA with 5 μg of random hexamers (Qiagen, Valencia, CA) for 10 min at 70°C and then with 200 U of Superscript II (Invitrogen, Carlsbad, CA) in the presence of 40 U of RNaseOUT (Invitrogen, Carlsbad, CA) for 2 h at 42°C. RNA was removed by RNase H (Invitrogen, Carlsbad, CA) treatment for 20 min at 37°C. Quantitative real-time PCR (qRT-PCR) amplification was performed separately with two sets of primers for each cDNA sample. Primers rcaC RT9 and rcaC RT10 were used to detect rcaC cDNA. Primers 23S RT1 and 23S RT2 were used to detect 23S rRNA cDNA as a control. A 25-μl PCR mixture containing 12.5 μl Platinum SYBR green qRT-PCR Supermix-UDG (Invitrogen, Carlsbad, CA), 0.5 μl of a 10 μM forward primer solution, 0.5 μl of a 10 μM reverse primer solution, 0.5 μl of a 500 nM fluorescein (Bio-Rad, Hercules, CA) solution, and 10 μl of a cDNA solution was amplified using an iCycler iQ multicolor real-time PCR detection system (Bio-Rad, Hercules, CA).

The PCR amplification program was 50°C for 2 min, followed by denaturation at 94°C for 10 min, 45 cycles of 94°C for 15 s, 50°C for 30 s, and 60°C for 30 s, and then 80 cycles of 55°C for 1 min with a 0.5°C increase in the temperature for every additional cycle that was performed to collect the melting curve data. The PCR products from one experiment were analyzed by agarose gel electrophoresis to ensure that a single product that was the correct size had been generated. In order to examine the efficiency of the qRT-PCR amplifications, standard curves were generated with a 1:10 serial dilution of sample cDNA for 23S rRNA and a 1:5 dilution for rcaC. A negative cDNA control, in which no Superscript II was added during the reverse transcription step, and another negative control, in which 10 μl of water was used instead of cDNA in the PCR amplification mixture, were added to each experiment to assess the degree of genomic DNA contamination, as well as the amount of contamination present in the reagents that were used. Eight independently isolated RNA samples from both RL- and GL-grown cultures were used in the qRT-PCR assay for analysis of both the rcaC and 23S RNA levels. The qRT-PCR results were analyzed using the method of Pfaffl (33).

Analysis of RcaC protein stability.[³⁵S]methionine was used to assess the efficiency of inhibition of protein synthesis by 20 μg ml⁻¹ chloramphenicol (CAP) in F. diplosiphon cells. Four 1.5-ml samples from a 50-ml culture of wild-type cells grown to an OD₇₅₀ of 0.5 in RL were used for each experiment. Three of these samples were treated with 20 μg ml⁻¹ CAP for 5 min, while no CAP was added to the fourth sample. After 5 min, 15 μCi of the Tran ³⁵S-Label metabolic labeling reagent containing [³⁵S]methionine (MP Biomedicals, Irvine, CA) was added to each tube. One sample containing 20 μg ml⁻¹ CAP was harvested immediately by centrifugation at 13,000 × g for 1 min to assess the extent of physical trapping of the ³⁵S label. The control containing no CAP and one sample containing 20 μg ml⁻¹ CAP were incubated in RL for 4 h, while the final sample containing 20 μg ml⁻¹ CAP was immediately moved to GL and incubated for 4 h. Cells from the three tubes were then harvested as described above. For all samples, supernatants were removed, and cell pellets were frozen in dry ice and then thawed three times before 50 μl of buffer B supplemented with 0.25 U/μl mutanolysin (Sigma-Aldrich, St. Louis, MO) was added. Samples were incubated for 30 min at room temperature and centrifuged at 13,000 × g for 1 min, and the supernatants were collected. Total protein was precipitated on ice for 30 min using 10% trichloroacetic acid. The precipitate from each sample was filtered onto a GF/C glass filter (Whatman, Brentford, Middlesex, United Kingdom), and total counts were recorded using an LS6500 scintillation system (Beckman, Fullerton, CA).

For analysis of RcaC stability, wild-type cells were grown in RL to an OD₇₅₀ of approximately 0.8. Four 50-ml cultures grown from a single starter culture were used in each experiment. One of these cultures was treated with 20 μg ml⁻¹ CAP and harvested immediately. Another culture was kept in RL for an additional 4 h without CAP before cells were harvested. The final two cultures were treated with 20 μg ml⁻¹ CAP and either kept in RL or switched to GL for an additional 4 h before cells were harvested. All cells were harvested by centrifugation at 5,000 × g for 5 min at 4°C. Total protein was then extracted from each culture and subjected to polyacrylamide gel electrophoresis and Western blot analysis to detect the RcaC protein (29). Equal protein loads were confirmed by Coomassie brilliant blue staining of equivalently loaded gels run in parallel with the gels used in Western blot analyses.

The kinetics of RcaC abundance changes are different from known CCA responses.The light-driven change in RcaC abundance might facilitate CCA-regulated alterations in the transcriptional activity and RNA accumulation from cpc2 and cpeBA, which change dramatically within 2 to 4 h after a light shift (30). Western blots were used to measure RcaC abundance over time after cells were shifted between RL and GL (Fig. 1A). Most changes in RcaC abundance occurred within 21 h after a change in the color of light in either direction, and the half-times for these changes were approximately 6 h for the shift from RL to GL and approximately 12 h for the shift from GL to RL (Fig. 1B).

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FIG. 1.

Kinetics of RcaC light-regulated abundance changes. (A) Western blot analyses of RcaC levels during growth under constant light conditions (0 h) or at various times after switches from RL to GL (upper blot) or from GL to RL (lower blot). The positions of molecular mass markers are indicated on the left. (B) Densitometric quantification of RcaC levels detected by Western blotting in cells grown in RL (filled squares), in GL (filled circles), in RL and then switched to GL (open squares), and in GL and then switched to RL (open circles) and harvested at 0, 4, 10, 16, 22, and 28 h after the light switch. The y axis indicates the amount of RcaC detected, which was normalized to the amount of RcaC under constant RL growth conditions. Each data point indicates the average for three independent replicates. The error bars indicate standard errors.

We then compared the kinetics of RcaC abundance changes shown in Fig. 1 to the timing of the shifts in the cellular PC/PE ratio after switches in the color of light during CCA. The kinetics of the shifts in PC and PE abundance were determined by extracting these phycobiliproteins and comparing their levels to the level of another phycobiliprotein, AP, whose abundance does not change significantly during CCA (13, 21), and the results were expressed as the ratio of PC to AP or the ratio of PE to AP. The complete CCA-driven shifts in PC and PE abundance took approximately 150 h, regardless of the direction of the shift in the light color (Fig. 2). The half-time for this process in both directions was close to 50 h. This is four to six times longer than the half-time for RcaC abundance changes during CCA (Fig. 1).

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FIG. 2.

Time course of the changes in the relative abundances of (A) PE and (B) PC in response to changes in the ambient light color. Levels of PC and PE were determined spectrophotometrically at various times after wild-type cells were shifted either from RL to GL or from GL to RL. The y axes indicate the cellular ratio of (A) PE to AP or (B) PC to AP. The experiments were repeated two or three times, and the error bars indicate the standard errors.

RcaC abundance changes require a functional RcaE photoreceptor.We have previously reported that RcaC is approximately six times more abundant in RL-grown cells than in GL-grown cells and that the abundance changes are eliminated if substitutions are made at either D51 or H316 of RcaC, two amino acids that are crucial for a normal CCA response (29). We further examined the mechanism(s) by which light color controls the level of RcaC by determining the abundance of RcaC in several different types of rcaE mutants.

The rcaE null mutant FdBk14 was grown in both RL and GL, and the RcaC abundance was checked (26, 39). In both light conditions, RcaC accumulated at intermediate levels relative to the levels found in wild-type cells grown in RL (Fig. 3A). This result suggested that RcaE could affect the differential accumulation of RcaC in RL and GL either through its physical presence or by its presumed ability to control the phosphorylation state of RcaC. We therefore examined RcaC levels in two additional types of rcaE mutants. The first mutant, the FdR9 mutant, was red during growth in RL and GL and contained an insertion between the H box and the ATP binding region of the histidine kinase domain. This insertion was predicted to lead to the synthesis of a truncated form of RcaE that dephosphorylated the Rca pathway in both RL and GL, since similar mutations in other histidine kinases result in the loss of kinase activity but retention of phosphatase activity (3, 9, 25). The second mutant, an RcaE site-directed mutant, RcaE-H430A, had an alanine substitution at the histidine at position 430 of RcaE, which is within the H box and is the likely site of phosphorylation during CCA signal transduction. This mutant was also red during growth in both RL and GL (Fig. 3B), indicating that RcaC was predominantly unphosphorylated in the cells (29). In both FdR9 and RcaE-H430A, the levels of RcaC were the same and relatively high in cells grown in RL and GL (Fig. 3A).

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FIG. 3.

RcaC levels in rcaE mutants. (A) Representative Western blot analysis of RcaC levels in RL- and GL-grown wild-type strain and rcaE null (FdBk14), rcaE truncation (FdR9), rcaE site-directed (pPLrcaEA430), and rcaC null (CR2) mutants. The positions of molecular mass markers are indicated on the left. Analyses were repeated either two or three times. (B) Representative whole-cell absorbance spectra of RL-grown (solid line) and GL-grown (dashed line) FdBk14 cells harboring the pPLrcaEA430 plasmid. PE and PC absorbance peaks are indicated.

RcaC abundance changes depend on the presence of the D51 and H316 residues themselves rather than on the CCA processes that they regulate.Previous work has shown that substitutions at both D51 and H316 lead to the production of forms of RcaC that are equally abundant during growth in RL and growth in GL (29). Based on this finding, together with the RcaE mutant results shown in Fig. 3, it is reasonable to hypothesize that the light regulation of RcaC abundance occurs via sensing of the phosphorylation state of RcaC, particularly at residues D51 and H316. However, the mutants are also not capable of normal CCA, making it possible that the loss of the light-regulated changes in RcaC abundance in the mutant cells were the indirect result of the loss of a CCA-regulated system that provided feedback control of RcaC abundance. This possibility was tested by producing C-terminally 3XFLAG-tagged versions of RcaC-D51N/H316Q and wild-type RcaC in wild-type cells. Whole-cell absorption spectral analyses of a number of transformed lines producing these proteins demonstrated that CCA, as measured by shifts in the PC/PE ratio during growth in RL versus growth in GL, functioned properly in all of these lines (Fig. 4A and 4B).

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FIG. 4.

RcaC abundance change requires D51 and H316. (A and B) Representative whole-cell absorbance spectra of wild-type (WT) cells transformed with (A) pPL3XFrcaC or (B) pPL3XFN51/Q316 and grown in RL (solid line) and GL (dashed line). PE and PC absorbance peaks are indicated. (C) Representative Western blot of wild-type cells transformed with either pPL3XFrcaC or pPL3XFN51/Q316 and grown in RL and GL. Anti-FLAG antibody was used to detect both of these forms of RcaC. (D) Quantification of 3XFLAG-tagged RcaC abundance, indicated by the ratio of the abundance of each protein in RL to the abundance of each protein in GL. The bars indicate the averages of six independent experiments, and the error bars indicate standard errors.

Transformed cells expressing either the wild-type or D51N/H316Q form of 3XFLAG-tagged RcaC were grown in RL and GL, and the levels of the proteins were detected using an anti-FLAG antibody (Fig. 4C and 4D). The cell lines producing wild-type RcaC-3XFLAG contained about 3.5-fold more of this protein in RL than in GL. Conversely, the cell lines producing RcaC-D51N/H316Q-3XFLAG contained approximately equal amounts of this protein during growth in RL and growth in GL. These results suggest that D51 and H316, rather than a downstream event that is part of the CCA response, are required for the light-mediated changes in RcaC abundance.

Both the D51, H316, and N576 residues and the abundance of RcaC regulate its activity.We assessed the importance of the levels of RcaC and its presumed phosphorylation state in the regulation of CCA by placing wild-type rcaC under the control of the copper-inducible petE promoter (6, 8) and transforming the resulting construct into the rcaC null mutant CR2. By controlling the copper concentration in the medium, we were able to manipulate the cellular level of RcaC in a light-independent fashion. Precise RcaC levels in the various transformed lines could be obtained consistently with specific concentrations of exogenously supplied copper (Fig. 5C).

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FIG. 5.

Effect of RcaC overexpression on CCA. (A and B) Representative whole-cell absorbance spectra for the CR2 mutant transformed with pPLPpetErcaC and grown in (A) RL and (B) GL with three different concentrations of copper (black line, 0 μM copper; dashed line, 0.3 μM copper; gray line, 1 μM copper). PE and PC absorbance peaks are indicated. Each scan is representative of the results for at least three independently transformed lines. (C) Representative Western blots showing RcaC abundance during growth in RL and GL for wild-type cells and cells of the CR2 mutant transformed with pPLPpetErcaC in the presence of three different concentrations of copper. Each Western blot analysis was repeated with at least two independently transformed lines.

For transformants expressing RcaC and grown in RL, increasing the RcaC level above the level present in wild-type cells had no discernible effect on the process of CCA (compare Fig. 5A and 5C to Fig. 4A). This suggests that in RL, the level of RcaC present in wild-type cells is sufficient to sustain the maximal CCA response. Because of the strength of the petE promoter, we were unable to produce RcaC at levels below those found in cells grown in RL and thus could not determine the effect of production of below-normal levels of RcaC in RL. Expression of RcaC in GL-grown cells at a level slightly above that found in wild-type RL-grown cells slightly inhibited the normal GL CCA response (compare Fig. 5B and C to Fig. 4A). Driving RcaC levels even higher strongly shifted the cells toward an RL phenotype, with decreased PE accumulation and increased PC accumulation (Fig. 5B and 5C). These results demonstrate that the lower abundance of RcaC present in wild-type cells grown in GL is required to obtain a normal CCA response in that color of light.

The RcaC levels in RL- and GL-grown cells were virtually the same in the absence of copper (Fig. 5C), but the CCA states were very different under these two conditions (Fig. 5A and 5B). This demonstrates that there must be differences in the activity of RcaC in these two light conditions that is controlled by a process that is independent of the abundance of this protein. These data, along with previous work, indicate that this additional process is the control of the phosphorylation state of the three amino acids in RcaC through which CCA is regulated, since cells expressing the RcaC-N51/Q316/N576 mutant at levels comparable to the levels observed in the absence of copper here (Fig. 5C) are not capable of undergoing CCA (29).

rcaC transcript levels are regulated by light.We have previously observed that RcaC abundance is greater during growth in RL than during growth in GL (29). We sought to determine whether this difference is partially or completely the result of corresponding changes in rcaC RNA levels under these two light conditions. RcaC abundance was quantified by Western blotting. Although the ratio of the RcaC level in RL to the RcaC level in GL was different in different experiments (data not shown), multiple measurements demonstrated that RcaC was 5.8 times more abundant in RL-grown cells than in GL-grown cells (Fig. 6A). We then performed qRT-PCR amplification of rcaC transcripts from RNA samples isolated from RL- and GL-grown cells. rcaC transcripts were slightly more than threefold more abundant in RL-grown cells than in GL-grown cells (Fig. 6B). Thus, the higher level of rcaC RNA in RL-grown cells than in GL-grown cells accounted for slightly more than one-half of the 5.8-fold difference in RcaC protein abundance measured under these two light conditions.

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FIG. 6.

(A) Relative RcaC abundance in RL- and GL-grown F. diplosiphon cells as determined by Western blot analyses. The RcaC protein level in GL-grown cells was defined as 1, while the RcaC level in RL-grown cells was expressed relative to the level in GL-grown cells. The averages for 29 Western blot analyses are indicated by the bars, and the error bars indicate standard errors. (B) Relative rcaC transcript abundance in RL- and GL-grown cells as determined by qRT-PCR amplification. The rcaC transcript level in cells grown in GL was defined as 1, and the level of rcaC RNA in RL-grown cells was expressed relative to the level in GL-grown cells. The bars indicate the averages for eight qRT-PCR experiments, and all values were normalized using 23S rRNA levels. The error bars indicate standard errors.

RcaC protein is less stable in GL than in RL.The differences in rcaC RNA levels (Fig. 6B) explained some, but not all, of the RcaC protein abundance differences (Fig. 6A) between growth in RL and growth in GL. Therefore, we examined the effect of growth in these two types of light on the stability of RcaC, utilizing CAP to inhibit protein synthesis. Although the method that we used has been reported to inhibit more than 90% of new protein synthesis (32), in our experiments an average of approximately 70% of new protein synthesis was inhibited, as measured by incorporation of [³⁵S]methionine (data not shown). Cultures were grown in RL and then either kept in RL or switched to GL. The cultures kept in RL were either treated or not treated with CAP, while those switched to GL were treated with CAP just prior to the switch. An RL-grown culture treated with CAP and then harvested immediately was also included as a control. Four hours after CAP treatment, cells were harvested, and RcaC protein abundance was measured using Western blot analysis. The RcaC levels in the cultures that were kept in RL and treated with CAP remained essentially unchanged (Fig. 7), suggesting that the RcaC protein is relatively stable under RL growth conditions. RcaC abundance in cells grown under the same light conditions and not treated with CAP also changed little over the same period. However, after 4 h, the level of RcaC protein found in cultures switched from RL to GL after CAP treatment was only approximately 65% of the level of RcaC protein found in cultures that were treated with CAP and kept in RL. These data strongly suggest that RcaC is less stable in cells that have been switched to GL from RL than in cells maintained in continuous RL.

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FIG. 7.

RcaC protein is less stable during growth in GL than during RL growth. Quantification of Western blot results provided relative RcaC levels in wild-type cells grown in the light conditions indicated. The bars indicate the means of four independent experiments. The error bars indicate standard errors of the means. CAP20, 20 μg ml⁻¹ CAP.