Proteomics Reveals a Role for the RNA Helicase ... - ACS Publications

ARTICLE pubs.acs.org/jpr

Proteomics Reveals a Role for the RNA Helicase crhR in the Modulation of Multiple Metabolic Pathways during Cold Acclimation of Synechocystis sp. PCC6803 John G. Rowland,† William J. Simon,† Jogadhenu S. S. Prakash,‡ and Antoni R. Slabas*,† † ‡

School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

bS Supporting Information ABSTRACT: One of the earliest and largest transcriptional responses that occur during exposure of Synechocystis sp. PCC6803 to cold is the induction of the crhR RNA helicase transcript. We show that crhR deletion results in failure to cold acclimate: there is reduced growth at 24 °C and marked impairment of growth at 20 °C. 2D-DIGE, using five biological replicates, was used to analyze the proteomic differences between the wild-type and ΔcrhR strains grown at (1) 34 °C and (2) following transfer from 34 to 24 °C (cold-acclimation). Sixteen significantly differentially expressed proteins were identified between the two strains grown at 34 °C. Forty-three distinct proteins were identified that responded to coldacclimation of the wild-type and 34 proteins for the mutant, with only 26 proteins common to both. A large proportion of the proteomic responses (76.5%) could not be predicted from published transcriptomic data. Only modest similarity is observed between proteomic and transcriptomic responses (r = 0.54
0.70). We propose functions for three previously hypothetical proteins. We suggest molecular targets for CrhR action and identify downstream regulated events in metabolism. KEYWORDS:

’ INTRODUCTION Biological organisms exist in an environment that is in a continual state of flux. The competitiveness of an organism is dependent on its ability to sense these environmental changes and modify its biochemistry in a timely and appropriate manner. The ability to adapt to a new set of environmental conditions can be enhanced by prior exposure to an intermediate set of conditions, a process known as acclimation. Acclimation is a cellular response to environmental change that results in a phenotypic alteration with no change in the genome. Acclimation is often initiated by a mild stress response involving transient physiological, biochemical and molecular perturbations. This is then followed by sustained alterations that are responsible for phenotypic adjustment to the new environment. Subsequent exposure to more severe stress is then less detrimental to the acclimated organism as has been shown, for example, for thermotolerance of photosystem II following acclimation to higher temperature.1 Temperature is a major environmental parameter with low temperature having a major impact on cellular productivity and growth rate. Cold acclimation has been studied in a number of model organisms including the gastrointestinal microbe Escherichia coli2 and the higher plant Arabidopsis thaliana3,4 among others. The wide variety of environmental niches and specific biochemistry of these organisms means that their cold acclimation mechanisms are likely to differ substantially. r 2011 American Chemical Society

The cyanobacterium Synechocystis sp. PCC6803 (hereafter Synechocystis) is a powerful model system for the study of stress responses in photosynthetic organisms. Cyanobacteria are aquatic photoautotrophic prokaryotes, an ancient ancestor of which is believed to be a progenitor of higher plant chloroplasts.5 The genome of Synechocystis has been completely sequenced and contains 3264 open reading frames (ORFs).6 The organism can be readily transformed and can incorporate exogenous DNA into its chromosome by homologous recombination.7 A DNA microarray is commercially available and the proteome of this organism is relatively small in terms of total number of distinct expressible proteins. Genome-wide transcriptional responses to both high and low temperature in Synechocystis have been reported previously.1,8,9 Low-temperature stress in this organism induces the expression of a large number of genes and a subset of these appear to be directly regulated by activity of the membrane-bound histidine kinase, Hik33.8 Low temperature-inducible genes included genes for ribosomal proteins, RNA binding proteins, subunits of RNA polymerases, NADH dehydrogenase subunits, acyl-lipid desaturases and proteins of yet unknown function. The crhR transcript, encoding an RNA helicase, is highly induced by low temperature stress in Synechocystis.8 Induction of Received: April 1, 2011 Published: June 17, 2011 3674

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Journal of Proteome Research this transcript occurs extremely rapidly and is one of the largest changes observed following low temperature stress. RNA helicases have been identified in many species from bacteria to human.10
13 These enzymes are intricately involved in the control of RNA secondary structure in an ATP-dependent manner. This activity is involved in the modulation of a wide range of cellular processes including translation,14 RNA decay,15
17 ribosome biogenesis12 and even translocation of proteins across membranes.18 A ΔcrhR knockout strain of Synechocystis has been generated by insertion of an antibiotic resistance cassette.9 The mutant grew as well as the wild-type at 34 °C, but exhibited marked growth impairment at a lower temperature of 24 °C. If the wildtype culture is transferred from 34 to 20 °C, there is a marked decrease in growth rate. This can be mitigated by acclimation of the 34 °C grown culture to 24 °C prior to exposure to 20 °C (this study). In the context of this manuscript, the cold acclimation changes we are concerned with are those that occur following transfer from 34 to 24 °C. Transcriptome analyses during coldacclimation of the ΔcrhR and wild-type strains have indicated that CrhR modulates the cold-inducibility of very few transcripts.9 Notably, deletion of crhR resulted in a reduction in the cold-inducibility of the groESL cotranscript and groEL2. CrhR was subsequently shown to be involved in maintaining RNA stability of these transcripts, preventing their degradation.9 However, no information is available on the proteomic changes that occur in either the wild-type or mutant under cold-acclimation conditions. Proteins are ultimately responsible for performing cellular metabolic processes and changes in their abundance are not always reflected at the transcriptional level.19,20 There remains some debate in this area as others have reported that a reasonable correlation does exist for the more abundant transcripts.21 It should be noted that these studies have focused on analysis of yeast cells and the findings are not necessarily universal to all organisms. The availability of proteomic data on the cold acclimation response in both the wild-type and mutant would complement the transcriptomic data. This could provide additional insight into the molecular biology and mechanisms underlying the cold acclimation process, possibly revealing events that are not predicted solely by a transcriptional analysis. In the current study we have analyzed the soluble proteomes expressed during cold-acclimation of both the wild-type and ΔcrhR strains, with judicious use of biological replication and consideration of statistical requirements. This has provided an indication of the major alterations to cellular metabolism that are associated with both successful and unsuccessful cold acclimation. Comparison of our data with publicly available microarray data has allowed us to suggest regulatory consequences of the involvement of CrhR in transcript- and protein- level regulation. We present a putative model of the events associated with acclimation to low temperature and the possible role(s) played by CrhR in this process.

’ MATERIALS AND METHODS Strains and Routine Culture Conditions

Glucose-tolerant Synechocystis sp. PCC 6803, was routinely grown photoautotrophically in BG-11 medium22 under constant illumination at 75 μmol photons m
2 s
1 with aeration by sterile air that contained 1% CO2. Maintenance cultures were grown at 34 °C. For maintenance of mutants, 25 μg/mL spectinomycin was included in the culture medium. Generation of the mutant defective in the RNA helicase CrhR has been described previously.9

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Cold Acclimation

Synechocystis cultures (500 mL) were subcultured at least three times at 34 °C in BG-11 medium, with light and gas supplied as for routine culture, to ensure they had achieved a steady-state that truly reflected these growth conditions. Cultures were then grown to an optical density (OD, A730) of 1.0 at which time experimental cultures were inoculated at a starting OD of 0.1. Experimental cultures were then grown to an optical density (A730) of 0.3
0.4 at which point they were transferred to 24 °C without disrupting illumination or aeration. Culture aliquots were taken for growth curve analysis and protein extraction at 0, 3, 7, 13, and 24 h after the transfer. Sample aliquots from control cultures that were not exposed to 24 °C were also taken. Experimental cultures of the mutant strain did not contain antibiotic. Growth and cell density were monitored by the A730 of cultures using a UV/vis spectrophotometer (Ultrospec 1100 Pro, GE Healthcare, www.gehealthcare.com). Triplicate measurements were taken for each of five independent biological replicates. Protein Sample Preparation and 2D-DIGE

Cells were harvested by centrifugation at 3000 g for 5 min. Cell pellets were then snap frozen in liquid nitrogen and stored at
80 °C prior to protein extraction. Cell pellets were resuspended in 500 μL of ice-cold Resuspension Buffer (25 mM TrisHCl, pH 8.0, 1 mM EDTA, 1 mM DTT). To this was added 25 μL of protease inhibitor cocktail (40 mg/mL, Sigma, P8465). Cells were mixed with an equal volume of washed and equilibrated 0.4 mm glass beads. Cell breakage was performed using a vortex mixer in 30 s bursts for a total of 5 min. Cells were placed on ice between bursts. Crude lysates were centrifuged at 2000 g for 5 min at 4 °C to remove unbroken cells. The supernatants were then further fractionated by centrifugation at 40 000 g for 30 min at 4 °C into soluble (supernatant) and membrane (pellet) fractions. Proteins from soluble fractions were collected by acetone precipitation23 and resolubilised in 100 μL of IEF sample buffer (9 M urea, 2 M thiourea, 4% CHAPS) containing 50 mM Tris-HCl at a pH of 8.5), adding further aliquots when necessary to ensure complete solubilization. Protein content was assessed by modified Bradford assay.24 2D-DIGE and gel imaging was performed as described previously,23 except that the second dimension separation was performed using the Dodeca Plus gel electrophoresis tank (BioRad) at a constant current of 10 mA/gel for 60 min followed by 15 mA/gel overnight at 15 °C until completion as assessed by migration of the dye-front. Each protein sample for analysis was minimally labeled separately with both Cy3 and Cy5 dyes, allowing for a fully dye-swapped experimental design. Preparative gels for spot picking contained 500 μg protein and were stained with SYPRO Ruby (also described previously23). Image Analysis

Gel images were analyzed using SameSpots software (Nonlinear Dynamics, U.K.). Our analysis workflow was fully datadriven and is described below. The effects of each data processing step were monitored throughout by principle components analysis. The set of images essentially contained two full copies of the experiment: one in the Cy3 channel and another in the Cy5 channel. In the initial exploratory phase, we identified spots that were significantly different between the two CyDye channels (as assessed by ANOVA, p e 0.05). These spots represent possible “dye effects” and were discarded from further analysis as they are a potential source of bias. For the experiment in each channel, we then proceeded to explore significant differences 3675

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Figure 1. Growth curve analysis of wild-type and ΔcrhR strains during cold-acclimation. Wild-type and ΔcrhR mutant cultures were grown at 34, 24, and 20 °C with continuous illumination of 75 μmol photons m
2 s
1. Cultures were inoculated to an OD (Abs730 nm) of 0.1 in liquid BG-11 medium and allowed to grow at 34 °C to an OD of 0.35
0.4. The cultures were then incubated at 24 °C for 3, 7, and 13 h followed by transfer to 20 °C. Both wild-type and mutant cultures were normalized to equal OD prior to transfer from 24 to 20 °C. BG-11 medium maintained at 24 °C was used for normalization. Growth was monitored over 72 h. (A) Temperature-dependence and the effect of cold-acclimation on growth of wild-type Synechocystis and (B) the ΔcrhR mutant strain. (C) Comparison of growth rates between wild-type and mutant cultures. (D) Representative photograph of wild-type and ΔcrhR mutant cultures taken 24 h post-transfer to 24 °C.

between sample groupings (0 h Wt vs Mutant, Wt 0 h vs 13 h, Mutant 0 h vs 13 h). Differential expression of a spot was declared as statistically significantly if and only if the change was significant in both channels, that is, if the statistical differences in one channel could be confirmed in the second. Power analysis was also performed at this point. Strict FDR criteria were set so that less than one false positive spot would be expected in each data set. The location of spots of interest on preparative gel images was determined using the built-in functionality of the SameSpots software package. Protein Identification by MALDI-TOF-TOF

Proteins of interest were robotically excised from preparative gels, robotically digested with trypsin and spotted onto MALDI target plates as described previously.23 MS/MS analyses were performed using a 4800 Proteomic Analyzer (Applied Biosystems, Foster City,

CA). TOF-MS spectra were obtained using automated data acquisition and processing controlled by Applied Biosystems 4000 series Explorer software, version 3.5. The instrument was operated in reflector mode, a mass range of 700
4000 m/z, a laser intensity of 3300 V and 1000 laser shots per spectrum. Acquired spectra were noise corrected, peak deisotoped and calibrated using the internal tryptic autolysis peaks at 842.500 and 2211.100 m/z. Up to eight precursor ions with the strongest ion intensity were selected for fragmentation and MS/MS analysis. This was performed using a 1 kV CID fragmentation method, collecting spectra from 4000 laser shots at an intensity of 3800 over the mass range. Peak lists of ion masses were generated from the calibrated and deisotoped MS and MS/MS spectra using GPS Explorer software version 3.6 (Applied Biosystems, Foster City, CA). Combined MS and MS/MS data were used to perform searches against all entries in the NCBI nr and CyanoBase 3676

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Journal of Proteome Research databases using the MASCOT search engine, version 2.2 (Matrix Science, Boston, MA). The following search parameters were used: digestion enzyme trypsin, single missed cleavage allowed, variable modifications of carboxymethyl cysteine and oxidized methionine, precursor mass tolerance of 50 ppm and fragment ion tolerance of 0.2 Da. A combined protein score greater than 76 (NCBInr) or 48 (CyanoBase) was considered a positive protein identification. Additionally, single peptide sequence based identifications from MS/MS only searches were included when the peptide score was at or above a threshold of 14 and the corresponding protein had been listed as the top hit in the combined searches.

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’ RESULTS AND DISCUSSION

occur early during cold acclimation. Proteomic analysis focusing on the early phase of the response should allow us to identify proteins involved in early metabolic events leading to the differential capacity of the strains to acclimate. To select an appropriate time-point for proteomic analysis 1-D SDS-PAGE analysis was performed on the wild-type strain grown at 34 °C following transfer to 24 °C (Supplementary Figure S1, Supporting Information). Samples were collected at 0, 3, 7, 13, and 24 h following transfer and the soluble fraction analyzed by SDS-PAGE. Differences in the soluble proteome of the wild-type strain were evident after 13 h of exposure to cold (Supplementary Figure S1A, Supporting Information). These changes may reflect the metabolic alterations that are associated with the development of cold tolerance. There was relatively little difference in the banding patterns between the 13 and 24 h timepoints. At least 8 bands change in abundance. These are highlighted in Supplementary Figure S1A, bands 1
8, and their intensities are plotted in Supplementary Figure S1B (Supporting Information). Cold-acclimation clearly involves induction of protein expression, up to a maximum of 3-fold for these bands, and occurs across the entire molecular weight range. From the plots, it is clear that expression of these bands is relatively stable, with most bands having reached maximal expression levels after 13 h, suggesting that the proteomic response is at or approaching a new steady-state at this point in time. A 2D gel-based analysis using the 13 h time-point is therefore likely to reveal the extent and identity of changes occurring in the soluble proteome.

RNA Helicase Encoded by crhR is Critical for Cold Acclimation of Synechocystis

Proteomic Experiment Design

Biomass productivity, or growth, is a simple and relevant measure of an organism’s competitiveness. We have investigated the effect of low temperature on the growth of both the wild-type and the ΔcrhR mutant strain. Incubation of 34 °C grown wildtype cultures at 24 °C for 3, 7, and 13 h exhibited enhanced growth rates when compared to the cultures that were directly shifted from 34 to 20 °C (i.e., without cold-acclimation at 24 °C). This data is presented in Figure 1A. The enhancement of growth was dependent on the duration of incubation, with longer incubation at 24 °C resulting in substantially better growth at 20 °C. An acclimation period of 13 h results in a culture density that reaches almost double that of the nonacclimated cultures. The wild-type is therefore clearly capable of successful cold acclimation. Such cold-acclimation enhanced growth was not observed in the ΔcrhR mutant cultures (Figure 1B). While 13 h acclimation of the wild-type resulted in a doubling of culture density, there is a negligible effect with ΔcrhR mutant cultures, demonstrating for the first time that this strain is defective in cold-acclimation. No distinguishable growth difference was observed between the wild-type and the ΔcrhR mutant cultures when grown at 34 °C (Figure 1C). However, at 24 °C there was a significant difference in the growth observed between wild-type and ΔcrhR cultures. There was a marked difference in the appearance of the wildtype and mutant cultures 24 h after transfer from 34 to 24 °C (Figure 1D), indicating that in some way the metabolism of the mutant strain is impaired. Little difference was observed in culture density of the strains up to 24 h post-transfer (Figure 1C), although clear distinction could be seen at 72 h. Previous transcriptome analysis, conducted using the same temperature regime, has revealed major differences in gene expression within 3 h of cold exposure.9 It is thus clear that metabolic and transcriptional changes

Requirements to perform adequate numbers of biological replicates and other considerations of experimental design have been highlighted in the proteomic literature.25 Sadly, while these are well-known, either the sheer number of replicates or the cost of reagents for experiments frequently precludes the application of the desired rigor in experimentation. To ensure that our data was robust and biologically significant we performed five independent biological replicates. These were generated for both the wild-type and ΔcrhR strains using a cold-acclimation period of 13 h, including matched controls that were not exposed to low temperature. We have used 2D-DIGE to identify the reproducible changes occurring in the soluble proteome, followed by protein identification by MALDI-TOF-TOF analysis. A full dye-swap design was used and the data analyzed using objective statistical criteria which included identifying potential sources of bias and assessing both the power of the current analysis and the number of replicates that would be required to derive a comprehensive data set at various fold-change thresholds. We have performed multiple comparisons between the wildtype and ΔcrhR strains at steady-state (34 °C) and under coldacclimation (13 h at 24 °C) conditions. Comparison of the proteomic content of the wild-type and ΔcrhR strains under steady-state conditions allows us to identify if there are any significant differences between the strains under these conditions. For each strain, comparison of cultures grown at 34 °C with those grown at 24 °C for 13 h allows us to identify changes associated with cold acclimation. Interrogation of the data sets collected also allow us to identify changes that occur in the wildtype but not in the mutant following transfer to 24 °C, taking into consideration any differences that may already exist between the two strains under normal growth conditions. Here we report the results of these comparisons. All reported changes are independent

Comparison of Proteomic Data with Publicly Available Transcriptomic Data

Data on the transcriptomic analysis of the cold-acclimation response of wild-type and ΔcrhR strains9 was downloaded from the KEGG Expression Database (http://www.genome.jp/kegg/ expression/). The data consisted of duplicate measurements of transcript abundance at 0, 20, and 60 min during a coldacclimation time-course experiment. Raw ratios were recovered for transcripts that corresponded to proteins identified in our proteomic analysis. The response profiles of these transcripts were then qualitatively compared to the responses observed for the matching proteins.

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of dye-effects, reproducible across all replicates and are significant in both the exploratory and confirmatory phases of the analysis. We have used the false discovery rate (FDR) approach to obtain our highest confidence data, expected to contain less than one false positive spot per data set. Data is also presented for spots that have been identified as statistically significant at a g 95% confidence level by ANOVA, as this is a standard approach in proteomic data analysis. These spots were significant in both copies of the experiment and are therefore unlikely to be false positives. Inclusion of all significant spots also reduces the chance of falsely declaring a protein as unique to any one data set and allows easier comparison across data sets. A summary of the overall differences observed across the three data sets and the number of spots picked and identified is given in Table 1. The locations of cold-acclimation responsive spots and interstrain steady-state differences on 2D gels are shown in Supplementary Figure S2 (Supporting Information). Protein identification data have been submitted to PRIDE26 (accession number 16471). Supplementary Table 1 (Supporting Information) contains data for all proteins of interest including expression changes, significance, confidence of identification and pI/molecular weight information. Any identified proteins annotated Table 1. Number of Significantly Different and Identified Spots in Each Comparison ΔcrhR vs

Wt cold

ΔcrhR cold

acclimation

acclimation

total Wt (34 °C) (34 to 24 °C) (34 to 24 °C) Significant Changes Significant and g1.2 Picked

169 167

30 26

121 118

99 98 62

103

18

74

Identified

84

16

64

52

Distinct Proteins

55

15

43

34

FDR Adjusted Changes

84

6

64

32

Picked

60

5

46

22

Identified

50

5

38

19

as hypothetical or of no known function were routinely submitted to BLASTP homology searches, as database annotations are not always as current as possible and reinterrogation of specific genes could give an indication of new function. Steady-State Proteome Analysis Reveals a Stress Phenotype for the ΔcrhR Mutant Strain Unrelated to Cold

2D-DIGE analysis of differences between the ΔcrhR mutant and the wild-type grown at 34 °C revealed 16 distinct protein spots, which corresponded to 15 distinct protein identities, that were differentially regulated above a fold-change threshold of 1.2 (Table 2). The locations of these spots on 2D gels are shown in Supplementary Figure S2A (Supporting Information), colored green if less abundant in the mutant and red if more abundant. Clearly there is very little difference between the wild-type and ΔcrhR strain proteomes under 34 °C steady-state growth conditions. The greatest increases were seen for certain isoforms of CysK (slr1842, spot 603), Gap2 (sll1342, spot 551) and TktA (sll1070, spots 221 and 227) which were increased by at least 1.4fold. A notable decrease was observed for the spot identified as Ppa (slr1622, spot 711, 1.4-fold). It is possible that these differentially expressed proteins/isoforms are responsible for the maintenance of a similar growth rate between the wild-type and the mutant. Some of these spots were present in trains on the gel. Although we have identified these other spots in the trains, no significant differential expression could be detected under steady-state growth conditions. Both the cysteine synthase CysK and the AhpC/TSA family protein Sll1621 (spot 728) were more abundant in the mutant than in the wild-type under 34 °C steady-state growth conditions. The amino acids cysteine and its metabolic derivative methionine are functionally important in many essential processes including electron transport and other redox systems as a result of the redox chemistry of the sulfur atoms they contain. The increased abundance of CysK in the mutant therefore suggests that redox systems maybe important for maintenance of growth in the mutant even under 34 °C steady-state growth conditions. This is

Table 2. Identified Protein Spots That Are Differentially Regulated in ΔcrhR versus Wild-type under 34 °C Steady-State Conditions

a

master spot no.

GeneID

GeneSymbol

name

164

sll1043

728

sll1621

458

slr1165

637 227

slr1051 sll1070

679

sll0807

cfxE

692

sll0395

218

sll0529

hypothetical protein

1.3

499

slr0394

pgk

Phosphoglycerate kinase

1.3

221

sll1070

tktA

transketolase

1.4

551

sll1342

gap2

Glyceraldehyde-3-phosphate dehydrogenase 2

603 711

slr1842 slr1622

cysK ppa

Cysteine synthase soluble inorganic pyrophosphatase

1.5a
1.4a

339

slr0452

ilvD

dihydroxyacid dehydratase


1.3a

676

slr1198

rehydrin


1.2a

670

slr0552

hypothetical protein slr0552


1.2a

pnp

change

polyribonucleotide nucleotidyltransferase

1.2

AhpC/TSA family protein

1.2

sat

sulfate adenylyltransferase

1.2

fabI, envM tktA

enoyl-[acyl-carrier-protein] reductase transketolase

1.2 1.3

pentose-5-phosphate-3-epimerase

1.3

phosphoglycerate mutase

1.3

1.4

Indicates spots that pass strict FDR criteria. 3678

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Table 3. Identified Proteins Associated with Cold-Acclimation of the Wild-type and ΔcrhR Strains master spot no.

unique to Wt

unique to ΔcrhR

GeneID

GeneSymbol

Wt change

name

ΔcrhrChange

functional categorya

578

•

sll0057

grpE

heat shock protein GrpE


1.3

A

759

•

sll0145

frr, rrf

ribosome releasing factor


1.3

A

577

•

sll0454

pheS

phenylalanyl-tRNA synthetase

1.3

A

alpha chain •

519 827 822

slr1356

rps1

30S ribosomal protein S1

sll0517

rbp1, rbpA

RNA binding protein

3b

838

1.3 •

408

slr0082

rimO

409

1.3

A

4.2b 2.2b

A

1.6b 3.9b

Ribosomal protein S12

3.2b

413 •

sll1261

tsf

Elongation factor TS

1.7b

slr1463

fus

elongation factor EF-G

3.7

2.3

157 156

2.8b 2.6b

2.3b 2

149

2.2b

1.7

096

1.7

650

A

3.8b

methylthiotransferase

160

080

A A

1.5 1.3


1.4

216 498

sll1818

rpoA

RNA polymerase alpha subunit

1.6b

1.4b

A

771

•

slr1251

cyp

peptidyl-prolyl cis
trans isomerase

2

A

754

•

slr1761

ytfC

FKBP-type peptidyl-prolyl cis
trans isomerase


1.5b

A

slr1720

aspS

aspartyl-tRNA synthetase

273

1.8b

272

1.6

270

1.5b

271

1.5b

824

slr2075

groES

10 kDa chaperonin (groES protein)

286

slr2076

groEL

60kD chaperonin 1

1.4 2b

1.5

1.3 1.3

A

1.6b

A

1.8b 1.6b

1.3

1.8b

1.7

164

1.6b

1.6b

163

1.4b

1.6b

279 278 177

sll1043

•

503 719

pnp

polyribonucleotide nucleotidyltransferase

slr1722

guaB

IMP dehydrogenase subunit

slr0185

pyrE

uridine 50 -monophosphate synthase

1.4

A

B,G

1.2 1.6

B

761

•

slr0676

cysC

adenylylsulfate 30 -phosphotransferase

1.5b

B

043 458

•

sll0370 slr1165

carB sat

Carbamoyl-phosphate synthase large chain sulfate adenylyltransferase

1.8 1.4b

B,C B,C

sll1234

ahcY

S-adenosylhomocysteine hydrolase

463

1.6

432

1.4

437

1.2

339

slr0452

ilvD

dihydroxyacid dehydratase

294

slr0657

lysC

aspartate kinase

284

C

1.3


1.2b


1.4

C

1.4b

1.4

C

1.3

605 607

sll0427

psbO

photosystem II Mn2+-stabilizing polypeptide psbO


1.9b
1.7b


1.3
1.3

D

779

sll1578

cpcA

phycocyanin a subunit


1.9b


1.5b

D

phycocyanin beta subunit


2.1

583

sll1577

cpcB

b


2b

D


1.7b

542 582

•

sll1580

cpcC1

phycobilisome rod linker polypeptide cpcC1

1.5

D

597

•

sll1579

cpcC2

phycobilisome rod linker polypeptide cpcC2


1.3

D

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Table 3. Continued master spot

unique to

unique to

no.

Wt

ΔcrhR

Wt GeneID

GeneSymbol

name

functional

change

ΔcrhrChange

840

•

sll1029

ccmK

CO2 concentrating mechanism protein

371

•

slr1643

petH

ferredoxin-NADP oxidoreductase

•

sll1994

hemB

porphobilinogen synthase

sll0807

cfxE

pentose-5-phosphate-3-epimerase

1.2

sll1070

tktA

transketolase

1.9b

1.5

1.7b 1.6

1.4

544 •

221

225

D E E

1.4

551

sll1342

gap2

Glyceraldehyde-3-phosphate dehydrogenase 2

1.4b

1.3

E

1.2b

554 •

sll1676

malQ

4-alpha-glucanotransferase

1.6

331

E

1.4

499 474

D D


1.2

223 227

330

1.5
1.3b
1.3

386 679

categorya

• •

692

slr0394

pgk

Phosphoglycerate kinase

1.5b

slr2094

glpX, fbpI

GlpX protein, fructose-1,6-/sedoheptulose-1, 7-bisphosphatase

1.7b

sll0395

phosphoglycerate mutase

1.3

E E


1.5b

E

644

sll1784

hypothetical protein sll1784


1.6


1.4b

F

532

slr0708

hypothetical protein slr0708


1.4


1.3

F

832

slr0923

hypothetical protein slr0923

1.4

F

slr0665

hypothetical protein slr0665

sll0529

hypothetical protein sll0529

1.4b

slr1649 slr1963

hypothetical protein slr1649 hypothetical protein slr1963


1.5b 1.7b

•

158 218

•

733 601

1.5b


1.5

slr2144

hypothetical protein slr2144


1.5

709

slr1852

hypothetical protein slr1852


1.7b

405

slr0965

dnaN

DNA polymerase III beta subunit

1.3

fabI, envM

enoyl-[acyl-carrier-protein] reductase

1.3b

rehydrin

1.1

668

•

b

F F


1.4 2b

F F


1.6b

F,G

F

(water-soluble carotenoid) 637

•

slr1051

676

•

slr1198

732 723

slr1516

sodB

superoxide dismutase


1.3
1.1

1.5b

G G G


1.2

G

a

Functional categories are as follows: A, Transcription, Translation and Protein Folding; B, Purine, Pyrimidine and Sulfur Metabolism; C, Amino acid Biosynthesis; D, Photosynthetic Electron Transport and Light Harvesting; E, Carbohydrate Metabolism; F, Hypothetical/Unknown; G, Others. b Indicates spots that pass strict FDR criteria.

further corroborated by the slightly elevated level of Sll1621, encoding a type II peroxiredoxin with glutathione-dependent peroxidase activity. This protein is believed to form part of an antioxidative stress system that is crucial for maintaining viability and aerobic phototrophic growth in both high and low light.27,28 The soluble inorganic pyrophosphatase Ppa and the hypothetical protein Slr0552 (spot 670) were less abundant in the mutant than in the wild-type under 34 °C steady-state growth conditions. Little is known about hypothetical protein Slr0552 but a BLAST search indicates that it may be a protein specific to cyanobacteria, as the only other ‘hit’ (low-confidence) was from Renibacterium salmoninarum ATCC 33209, the causative agent of bacterial kidney disease in salmonid fish. More is known about the pyrophosphatase Ppa which is an essential protein in bacteria,29 being responsible for the cleavage of inorganic pyrophosphate (PPi) to orthophosphate (Pi). This makes a number of vital anabolic reactions such as DNA/ RNA synthesis and tRNA charging thermodynamically irreversible. It has also been reported that the fidelity of DNA synthesis is

reduced with lower PPi concentrations or higher pyrophosphatase activity.30 The decreased abundance of this protein in the ΔcrhR strain, suggesting the accumulation of polyphosphate, might indicate that there is a dysregulation of certain anabolic reactions or that higher fidelity DNA synthesis is required in the mutant strain for maintenance of growth under 34 °C steady-state growth conditions. The remaining proteins that are differentially expressed between the wild-type and mutant strains under 34 °C steady-state growth conditions are discussed below in the context of coldacclimation as they are also present in the cold-acclimation response of at least one of the two strains. A Large Number of Proteomic Changes Associated with Diverse Metabolic Functions Occur during Cold-Acclimation of the Wild-type

Exposure to cold results in a number of physicochemical constraints on cellular function. Lipid bilayers undergo transition 3680

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Journal of Proteome Research from a liquid crystalline state to a gel state (reduction in fluidity), general protein synthesis is transiently inhibited and the production or import of compatible solutes is induced. Metabolic activity is also reduced as a result of both the inherent effect of temperature on enzyme activity and also the lower growth rate requiring less metabolic capacity to sustain it. While these general features are known, the identity and interplay of components specifically involved in the cold-acclimation response of wildtype Synechocystis is less well characterized, particularly at the protein level. 2D-DIGE analysis of the cold-acclimation response of wildtype Synechocystis revealed significant changes (at least 1.2 fold) for 118 spots, of which 74 spots were selected for further analysis. The remainder were either not clearly resolved on the preparative gels or were considered to be of too low abundance to give a high likelihood of identification. Synechocystis contains a number of autofluorescent proteins, obscuring spot boundaries and making it difficult to determine accurate picking coordinates for these spots and their neighbors, so they were excluded from our pick list. Significant changes are highlighted on the master gel image shown in Supplementary Figure S2B (Supporting Information), with upregulated spots colored green and down-regulated spots colored red. Following in-gel digestion and MALDI-TOF-TOF analysis, the picked spots resulted in the identification of 43 distinct proteins. These proteins were classified into functional categories broadly based on the KEGG annotation. The fold-change and functional annotation for each identified protein is shown in Table 3. Clearly, the proteins that alter in abundance during the cold acclimation response are associated with multiple metabolic functions. As these pathways involve multiple components, consideration of the role of individual proteins is required. At the time of writing, literature searches performed for each ORF ID and various keywords (i.e., cold, acclimation, cold acclimation, cold shock) revealed that none of these proteins, except GroES and GroEL, have been previously associated with cold acclimation in Synechocystis. The dominant feature of the wild-type cold-acclimation response was the substantial proportion of proteins associated with “transcription, translation and protein folding”. All of the proteins in this category were elevated during cold-acclimation, with the exception of the heat shock protein GrpE (Sll0057, spot 578), ribosome releasing factor Rrf (Sll0145, spot 759) and the peptidyl-prolyl cis-trans isomerase YtfC (Slr1761, spot 754). The induction (1.6-fold) of RpoA (spot 498), the alpha subunit of RNA polymerase, suggests an increased level of the RNA polymerase holoenzyme that may serve to compensate for reduced catalytic activity at lower temperatures. The most substantially induced protein was RNA binding protein (Rbp1, spots 827 and 838, up to 3-fold), which is known to be a coldregulated transcript and is believed to be involved in regulating RNA structure.31 This might provide a mechanism for improving translational efficiency by maintaining transcript availability and accessibility by the ribosomes under conditions of low temperature. Polyribonucleotide nucleotidyltransferase (Pnp, Sll1043, spots 163, 164 and 177), involved in RNA degradation, was also substantially induced (up to 1.8-fold) and probably serves to eliminate transcripts with cold-induced excessive secondary structure.32 This would effectively recycle ribonucleotides for transcription and also help to reduce the negative impact of malformed RNA molecules on the translation process. Translation is also clearly affected by cold as elongation factors TS (Tsf, spot 650) and G (Fus, spots 096, 149, 156, 157, 160

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and 216) were substantially induced during cold-acclimation (1.7-fold and up to 3.7-fold, respectively). This indicates that translational efficiency is negatively affected by cold and suggests that these proteins may serve to mitigate this effect. Interestingly, hypothetical protein Slr0923 (spot 832, 1.5-fold induction) was found to have substantial homology to PSRP-3 of Cyanothece sp. PCC 7822 (81% identity, 90% positive, 0% gaps). It has been suggested that this 30S ribosomal subunit protein might be involved in light-dependent translation regulation.33 There was also a small downward change (1.3-fold) in the abundance of ribosome releasing factor (Rrf, spot 759), involved in ribosome recycling at the termination of polypeptide synthesis,34
36 although whether this change has a substantial biological impact is unclear. Protein structure and function are also affected by reduced temperatures, as stalled ribosomes result in the formation of truncated proteins and intact protein conformations are altered. These effects can be mitigated by the expression of chaperonins and peptidyl-prolyl isomerases.37
39 The major chaperone systems in bacteria are the DnaK and GroEL mechanisms. These mechanisms appear to act sequentially. Nascent polypeptides interact with the peptidyl-prolyl isomerase trigger factor, are processed by DnaK and DnaJ using ATP and released after GrpE-mediated nucleotide exchange as fully or partially folded proteins. Further isomerization of partially folded proteins and prevention of protein aggregation can be achieved by the GroEL and GroES chaperonins.37,40,41 GroEL has also been shown to act in the cotranslational folding of polypeptides as they exit the ribosome.42 In addition, GroEL is able to fold proteins in the absence of GroES.43 In our proteomic data set on wild-type cold acclimation, GrpE (spot 578) is slightly down-regulated, suggesting either that the immediate response to partially folded proteins is largely complete at the time-point sampled or that the rate of synthesis of new proteins is reduced. It is also clear that the stoichiometric relationship between the level of GroEL (Slr2076, spots 278, 279 and 286, up to 2-fold induction) and GroES (Slr2075, spot 824, 1.4-fold induction) is altered. This might suggest that under cold conditions, the functional requirement for the GroEL-GroES chaperonin system is skewed away from the classical description as a bipartite system and toward a mechanism favoring the action of GroEL alone. This is somewhat unexpected as they are present in an operon in the genome, suggesting the operation of additional nontranscriptional regulatory influences for these proteins. The situation is somewhat complicated as GroES is only represented by a single spot (spot 824), the neighboring spots (832 and 838) are different proteins. GroEL on the other hand is part of a spot train, the components of which have been previously characterized as GroEL. The major spots in the GroEL train are spots 278, 279 and 286 and these all increase in abundance. Interestingly, our data set also reveals the apparently anticorrelated expression of two peptidyl-prolyl cis-trans isomerases (PPIase), responsible for assisting protein folding during translation; Cyp (slr1251, spot 771) is induced 2-fold while YtfC (slr1761, spot 754) is repressed 1.5-fold. This might suggest a shift in the translation efficiency of specific target proteins as PPIases generally possess distinct substrate specificities.44 Peptide bonds involving proline residues can adopt both cis and trans conformations and can therefore result in distinct protein conformations. As a consequence, PPIases are believed to play important roles in protein folding 3681

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Journal of Proteome Research and maintaining appropriate protein conformation. In photosynthetic organisms, PPIase have been reported to be involved in import of Rieske protein into the thylakoid membrane,45 accumulation of the PSII supercomplex,46 and insertion of the manganese-stabilizing protein, PsbO, into PSII.47 However, proteomic analysis of Arabidopsis lines bearing T-DNA insertions in a number of PPIase has shown that there was no significant difference in major thylakoid proteins between wild-type and mutant lines under standard as well as high light and cold stress growth conditions.48 Clearly, the function of these PPIase is not limited to protein folding and the nature of their involvement in the cold-acclimation response of Synechocystis, and indeed other photosynthetic organisms, warrants further investigation. Two tRNA synthetases were also observed to increase during cold acclimation of the wild-type, with the expression of aspartyltRNA synthetase (slr1720, spots 270
273) induced 1.8-fold and phenylalanyl-tRNA synthetase (sll0454, spot 577) only slightly induced at 1.3-fold. Reasons for this could be (i) an increased demand for these specific residues to be incorporated into new protein or that (ii) the cellular pools of aspartate and phenylalanine could be depleted during cold acclimation, with the synthetases being induced to compensate for the lower concentration of these metabolites. The latter explanation is consistent with the presence in our data set of five additional proteins involved in “amino acid biosynthesis” (see Table 3 for identities and fold-changes). Two of these additional proteins, CarB (sll0370, spot 043) and Sat (slr1165, spot 458), are also linked to “purine and pyrimidine biosynthesis”, a category which also contains PyrE (slr0185, spot 719) and CysC (slr0676, spot 761). Together this indicates that the capability for nucleotide production and recycling is important during cold-acclimation. The presence of these proteins in our data set is indicative of an increased demand for nucleotide and an alteration in the balance of amino acid availability, consistent with the requirements for protein synthesis described above. Overall, the data discussed so far is consistent with an increased demand for protein synthesis and suggests that this demand is met at all levels of regulation (transcriptional, translational and post-translation). As might be expected from a photosynthetic organism, proteins involved in “photosynthetic electron transport and light harvesting” are cold-acclimation responsive. Light harvesting by Synechocystis is achieved by the phycobillisome, a pigmented multiprotein complex that mediates the transfer of broadspectrum light into the photosystem reaction centers. The phycobillisome contains an allophycocyanin core (ApcA
F) and phycocyanin rods (CpcA
G). The relative amount of energy delivered to each of the photosystems is regulated by “state transitions”. Photosystem II (PSII) is responsible for the watersplitting reaction, generating protons for ATP synthase, electrons and molecular oxygen. This reaction is dependent on the presence of manganese, which is maintained by the manganese-stabilizing polypeptide PsbO. The expression of proteins involved in photosynthetic electron transport and light harvesting was almost universally repressed in the wild-type during cold-acclimation, with most of the identified components being part of the phycobillisome. The phycocyanin alpha (CpcA, sll1578, spot 779) and beta (CpcB, sll1577, spots 542 and 583) subunits, forming the rods of the phycobillisome, were virtually identically repressed about 2-fold. Two rod linker polypeptides, CpcC1 (sll1580, spot 582)

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and CpcC2 (sll1579, spot 597) were also present in our data set. Interestingly, despite these proteins being encoded in an operon with CpcA and CpcB,6,49 CpcC1 was up-regulated (1.5-fold) while CpcC2 was down-regulated (1.3-fold) under cold-acclimation conditions. It may be important to note that cpcC1 lies at the distal end of the operon sequence and the apparent anomalous expression of the protein could be the result of post-transcriptional regulation. It is possible that a number of distinct functional subpopulations of phycobillisome complex exist within the cell. The change in the relative ratio of CpcC1 to CpcC2 suggests remodelling of the phycobillisome structure and/or spectral properties.50,51 Degradation of the phycobillisome has also been observed under conditions of nutrient-limitation and may serve as a nitrogen-source.52 Interestingly, a BLASTP search using the hypothetical protein Slr1649 (spot 733) query sequence identified this protein as belonging to the CpeT/CpcT family. This protein family is restricted to cyanobacteria, cryptomonads and plants. In Synechococcus sp. PCC 7002, CpcT encodes a bilin lyase that is responsible for the attachment of phycocyanobilin to the βsubunit of phycocyanin.53 Indeed, the top-scoring hit in the BLAST search was bilin lyase of Synechococcus sp. PCC 7002 (54% identity, 70% positive, 1% gaps). If performing the same function in Synechocystis, this would again point to alteration of light harvesting capacity during cold acclimation. The manganese-stabilizing polypeptide, PsbO (sll0427, spots 605 and 607), was also repressed almost 2-fold during coldacclimation. PsbO is synthesized outside of the thylakoid and subsequently imported into the thylakoid lumen, where it functions to stabilize the oxygen-evolving manganese cluster of PSII. A reduced level of soluble PsbO in the extra-thylakoid compartment is indicative of a possible lower demand for that protein and consequently a lower PSII activity. As PSII is the main source of production of reactive oxygen species (ROS), the decrease in the abundance of the ROS-detoxifying superoxide dismutase (SodB, discussed later) would be consistent with lower PSII activity. Although there may be a reduced demand for photosynthesis, it is very obvious from our data set that “carbohydrate metabolism” is strongly influenced by cold, with at least six proteins associated with this process all being induced. Specific pathways that come under this category include the pentose phosphate pathway, glycolysis/gluconeogenesis and starch and sucrose metabolism as well as including the process of carbon fixation by the Calvin cycle in photosynthetic organisms. The pentose phosphate pathway, Calvin cycle and gylcolysis/gluconeogenesis pathways share a number of enzymes. For example Fbp1, GlpX and Fda are common to all three pathways, TktA and CfxE are involved in both the Calvin cycle and pentose phosphate pathway, and Gap2 and Pgk are involved in both the Calvin cycle and glycolysis/gluconeogenesis. The Calvin cycle consists of 10 enzymes, of which we have identified five as differentially expressed during cold-acclimation of wild-type Synechocystis. The Calvin cycle is the only pathway to which all of these identified proteins could be matched. All of these proteins were up-regulated, which may reflect an attempt to optimize the catalytic activity of the Calvin cycle following reduction in temperature. The up-regulated hypothetical protein encoded by sll0529 (spot 218) was found to possess substantial homology (80% identity, 88% positive, 0% gaps) to the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase of Cyanothece sp. PCC 8802, suggesting that this may be a second transketolase in 3682

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Journal of Proteome Research Synechocystis. The only protein involved in carbohydrate metabolism that could not be mapped on to the Calvin cycle is the protein encoded by sll1676, 4-alpha-glucanotransferase (MalQ, spots 330 and 331), which is associated with the degradation/ utilization of glycogen stores. This indicates that, under cold conditions, Synechocystis is unable to photoautotrophically satisfy demand for carbon and/or ATP and must make use of endogenous carbon stores. Proteins identified as belonging to the “Other” category included the repression of water-soluble carotenoid protein (spot 709) and superoxide dismutase (SodB, spots 723 and 732), and the induction of enoyl-ACP reductase (spot 637), the beta subunit of DNA polymerase III (spot 405) and, to a small extent, rehydrin (spot 676). Although the function of rehydrin in Synechocystis is unclear, it has been speculated that it may be involved in protection from continuous illumination and assisting functional assembly of the photosynthetic machinery.54 It is not clear whether the small change in abundance for this protein plays a significant biological role in the cold-acclimation response. Superoxide dismutase, encoded by sodB (spots 723 and 732), is the only superoxide scavenging enzyme present in Synechocystis6,55 and is responsible for detoxification of reactive oxygen species that are inevitably produced by oxygenic photosynthesis. This enzyme is also light regulated.56 The lower abundance of this protein in our data set suggests that the intracellular level of ROS is reduced under cold conditions and supports our earlier suggestion that photosynthetic capacity may be reduced. The water-soluble carotenoid protein that is similarly involved in photoprotection57,58 was also repressed (1.7 fold, spot 709). Light triggers a shift from an orange form to a red form, which induces energy dissipation by the phycobillisome and results in reduced energy arriving at the reaction centers.59,60 Reduced abundance of this protein under cold conditions suggests that whatever light can be harvested by the phycobillisome would be more efficiently directed to the reaction centers. DnaN (spot 405) was slightly induced under cold conditions. This protein is the beta subunit of DNA polymerase III forming the sliding clamp and is important for replicative DNA synthesis. DnaA is important in the initiation of DNA replication and, in E. coli, DnaN regulates the chromosomal replication cycle by interacting with and negatively regulating DnaA activity.61,62 Clearly, the increased abundance of DnaN under cold conditions has consequences for cell division. It could be that the increased abundance observed is an indicator of successful cold acclimation allowing increased cell growth rate after mitigation of the original cold shock. Alternatively, it could represent the strict control placed on DNA synthesis to limit cell growth at a level that metabolic processes can support. Enoyl-ACP reductase (FabI, spot 637), a component of the type II fatty acid synthase responsible for de novo fatty acid synthesis, was induced with a high confidence during cold acclimation. Fatty acid synthase is a dissociable complex. It is possible that the remaining components of fatty acid synthase, all of which are soluble proteins, also exhibit differential expression but that they were simply not picked or identified in this study. That seems unlikely as they should be expressed at a similar level of abundance. The other possibility is that expression of FabI is specifically regulated separately from that of other fatty acid biosynthetic enzymes, altering the stoichiometry of the complex. In E. coli, this enzyme can utilize both NADH and NADPH as cofactor, albeit with differing reaction kinetics,63 and the specific

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regulation of FabI might allow Synechocystis to compensate for a deficiency in fatty acid biosynthesis that could be caused in the event of an altered NADH:NADPH stoichiometry. It is known that both de novo fatty acid biosynthesis and desaturation play critical roles in low-temperature growth, respiration and photosynthesis.64,65 It should be noted that one would not expect to see any of the acyl-lipid desaturases of Synechocystis in our proteomic data set. These enzymes are membraneassociated/bound, and we have focused our analysis on the soluble fraction only. The diversity of the functional roles of the proteins associated with cold acclimation of the wild-type is consistent with there being a substantial rebalancing of metabolism. Also, the altered ratios of PPIase isoenzymes (involved in transcription, translation and protein folding) points to specific members of gene families being more important under cold acclimation conditions. Modified Proteomic Response to Cold Is Exhibited by the ΔcrhR Mutant Strain Relative to the Wild-type

Following our analysis of the wild-type cold-acclimation response, we then proceeded to analyze the effect of coldacclimation on the proteome of the ΔcrhR strain. We observed 98 spots that changed in abundance by more than 1.2-fold, of which 62 spots were picked from a preparative gel. Significant changes are highlighted on the master gel image shown in Supplementary Figure S2C (Supporting Information), with upregulated spots colored green and down-regulated spots colored red. This resulted in the identification of 34 distinct proteins from a total of 52 identifications (Table 3). These proteins were classified into functional categories based on the KEGG annotation. As would be expected, the cold-acclimation response of the mutant included many of the same proteins as the wild-type response. This indicates that the general features of the wild-type response are retained in the mutant. However, a number of potentially crucial differences are evident: 8 proteins are unique to the mutant cold-acclimation response while 14 proteins that were involved in the wild-type acclimation response are absent from the mutant response. These proteins are indicated as “Unique in Wt” and “Unique in ΔcrhR” in Table 3. In addition to these qualitative differences in the cold-acclimation response between the two strains, a number of quantitative differences exist in the magnitude of the responses of the set of common proteins (Table 3). The possible functional consequences of all differences between strains, including unique proteins, are discussed below. Similarly to the wild-type response, all proteins in the category of “transcription, translation and protein folding” were upregulated during cold-acclimation of the mutant. However, this category included two additional proteins that were not observed to change during wild-type cold-acclimation. These were RimO (spots 408, 409 and 413) and Rps1 (spot 519), both associated with the ribosome. A relatively large number of proteins in this category that were previously seen to change in the wild-type did not do so in the mutant. These included GrpE, Tsf, Rrf, PheS, and both PPIases Cyp and YtfC. The appearance of RimO, involved in ribosome modification (methylthiolation),66 and Rps1, a core structural component of the ribosome essential for translation,67 in the mutant cold-acclimation data set suggests an increase in ribosome biogenesis and activity relative to the wild-type response. This is further corroborated by the nonresponsive expression of Rrf and Tsf, indicating an increased rate of ribosome recycling. The absence of PheS in the mutant response indicates either that phenylalanyl-tRNA is in less 3683

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Journal of Proteome Research demand or that the cell has diminished capacity to charge this specific tRNA. Interestingly, the nonresponsive nature of GrpE and the two PPIases Cyp and YtfC suggests a major defect in the protein folding machinery of the mutant. It is notable that the expression of RNA binding protein, Rbp1 (spots 822, 827 and 838), which is cold-responsive in both the mutant and wild-type, is induced to substantially higher levels in the mutant. This protein might serve to increase the transcript accessibility of ribosomes under cold conditions. The remaining proteins in this category are common to the wild-type response but exhibit a slightly lower magnitude of change in the mutant. This indicates a possible reduction in translation rate in the mutant although it is not clear whether this is a cause or effect of translational deficiency and requires further testing. The categories of “purine, pyrimidine and sulphur metabolism” and “amino acid biosynthesis” were relatively similar to the wild-type response. IMP dehydrogenase (GuaB, spot 503) was unique to the mutant response and, in E. coli, catalyzes the ratelimiting step in guanine nucleotide biosynthesis and is essential for maintaining the normal ATP and GTP pool sizes.68 This suggests that these pool sizes may be imbalanced in the mutant during cold-acclimation. Notably, both CysC and CarB were not cold-responsive in the mutant but were strongly induced in the wild-type. CarB is responsible for the synthesis of carbamoyl phosphate, a key intermediate in the biosynthesis of both pyrimidine nucleotides and arginine, while CysC is involved in sulfur metabolism and as such is important for the synthesis of the sulfur-containing amino acids cysteine and methionines. This again points to the likely importance of redox chemistry, mediated via sulfur compounds, during cold-acclimation as the mutant is deficient in aspects of their synthesis. There is also some evidence to suggest the potential differential involvement of Sat protein isoforms between the two strains, as different spots corresponding to this protein on the gel were altered in response to cold. Within the category of “photosynthetic electron transport and light harvesting”, the phycobillisome components CpcC1 and CpcC2 were not cold-acclimation responsive in the mutant. Down-regulation of CpcA (spot 779) was also more pronounced in the wild-type while the fold-change for CpcB (spot 583) was comparable between strains. The differential response of proteins encoded on the cpcABCD operon (namely CpcA, CpcB, CpcC1 and CpcC2 in this data set) is particularly interesting as it suggests that CrhR may exhibit cistron-specific regulatory influence. Down-regulation of PsbO (spots 605 and 607) was lower in the mutant compared with the wild-type. This lower response could be the result of a reduced rate of insertion of this subunit into PSII by PPIases under cold conditions, as discussed earlier, rather than any transcriptional or translational regulation. These changes are accompanied by mutant-specific down-regulation of PetH (ferredoxin-NADP oxidoreductase, involved in electron transport around PSI, spots 371 and 386), and HemB (porphobilinogen synthase, involved in chlorophyll metabolism, spot 544), and induction of the carbon dioxide concentrating mechanism protein CcmK (spot 840). CcmK is a structural component of the carboxysome microcompartment where CO2 is concentrated to enhance Rubisco activity. CcmK homologues are not always regulated in stoichiometric amounts and variability in the composition of the carboxysome shell is believed to play a role in the response to environmental changes.69,70 Clearly changes are occurring for a number of components involved in carbon dioxide concentration and photosynthetic electron transport systems.

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Alterations in photosynthesis and light harvesting associated proteins in the mutant might be expected to have an influence on “carbohydrate metabolism”. In this category, the mutant exhibits a slightly reduced response to cold for proteins that are also present in the wild-type response (TktA: spots 221 and 223, Gap2: spots 551 and 554, Pgk: spot 499). The mutant response is also lacking in the differential expression of CfxE, GlpX, MalQ and the hypothetical protein Sll0529 that may be a second transketolase. A relative deficiency in both GlpX and CfxE is likely to have a negative impact on the productivity of both carbon fixation and the pentose phosphate pathway. It is possible that these deficiencies (in GlpX and CfxE) are linked to induction of the carbon dioxide concentrating mechanism protein that we have observed. If carbon compounds are not made in sufficient quantity, this may be sensed as carbon limitation resulting in induction of the carbon dioxide concentrating mechanism. Perhaps the most interesting difference in carbohydrate metabolism between the two strains is the mutant-specific down-regulation of phosphoglycerate mutase (sll0395, spot 692). This protein occupies a critical metabolic location effectively controlling carbon flux from glycerate-3-phosphate, at the intersection of carbon fixation and glycolysis/gluconeogenesis, ultimately to pyruvate. The mutant-specific down-regulation of this protein indicates that fixed carbon is redirected toward gluconeogenesis and the pentose phosphate pathway rather than the TCA cycle and energy production via glycolysis. This may allow the increased production of compatible solutes that serve to protect the cell from changes in osmotic potential that are a consequence of low temperature. The major compatible solute for Synechocystis is glucosylglycerol, which is formed by the reaction of ADP-glucose with glycerol-3-phosphate.71,72 Additionally, the lack of carbon entering the TCA cycle would limit the production of precursors for fatty acid synthesis and various amino acids. The nonresponsiveness of MalQ to cold in the mutant suggests that this strain is relatively impaired in its ability to mobilize glycogen stores and this, coupled with reduced photosynthesis and the redirection of carbon flux is likely to contribute substantially to the impaired growth of the mutant strain under cold conditions. The slightly lower response observed for water-soluble carotenoid protein (spot 709) and SodB (spot 732) in the mutant response may well reflect a reduced ROS load on the cell as a result of reduced photosynthetic electron transport. The slight enhancement in the response of DnaN (spot 405) to cold conditions relative to the wild-type suggests that this protein is acting as a “brake” on DNA synthesis and cell growth rather than being an indicator of successful acclimation. In the mutant, FabI and rehydrin are not cold-responsive. This indicates that the mutant may not be capable of sufficient de novo fatty acid synthesis to allow it to appropriately regulate membrane physical parameters. The level of rehydrin protein in the mutant is already lower than that of the wild-type under standard conditions. The potential functions for this protein, namely protection and functional assembly of photosynthetic machinery, have been discussed earlier. An intrinsically lower level of this protein, combined with failure to induce under cold conditions, may contribute to the disruption of photosynthetic electron transport in the mutant and consequent growth deficiency. Mutation of the crhR gene results both in qualitative and quantitative differences to the wild-type response. Comparison with available transcriptomic data would allow one to determine 3684

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Figure 2. Proteins that respond during cold-acclimation of wild-type and ΔcrhR Synechocystis. Proteins that are common to the response of both strains or unique to either strain are shown. The dashed line indicates an FDR cutoff that results in data sets with a likelihood of containing less than 1 falsepositive spot.

how many of these protein alterations could be predicted from array studies. Comparison with Transcriptome Data Reveals Limited Correlation between Transcript and Protein Responses

Transcriptomic data is often used as a proxy for protein expression data that has not been directly observed in proteomic investigations. Failure to observe certain proteins can be the result of a number of factors, including low abundance, protein isoelectric points and the nature of the sample preparation procedure. Transcriptomic data could be used to infer the response of additional, unobserved, protein components involved in metabolic/signaling pathways. Transcriptomic data could also be used to help differentiate between transcriptional and translational effects of regulatory proteins. While transcriptomic approaches are capable of quantifying responses for all transcripts, they cannot identify post-translational modifications (PTMs) that can be of great importance in metabolic regulation and signal transduction. Transcriptomic data on the cold-acclimation response of wildtype Synechocystis and ΔcrhR strains is publicly available.9 That data set was generated using the CyanoCHIP commercial array platform and analyzed the transcriptome of the wild-type and the ΔcrhR strains at 20 and 60 min after transfer from 34 to 24 °C. When using the CyanoCHIP array platform, it is standard practice to only consider ORFs with expression ratios greater than 2 as potentially differentially expressed8 since these changes have been found to be robust as validated by qRT-PCR experimentation. In the following discussion, we define transcriptional responses with a fold-change of 2 or more as “significant” and those below it as “non-significant”. We filtered the transcriptomic data to include only those transcripts for which we had proteomic data on the cold acclimation response, giving a total of 51 proteins matched to transcripts. On this basis, 26 identified proteins could be matched to significant transcript changes, while 25 protein changes were matched to nonsignificant transcript changes (Supplementary Table 2, Supporting Information). We then proceeded to examine the degree of similarity between transcript and protein responses.

Interrogation of the 26 significant transcript changes, in comparison to our proteomic data, revealed that only 12 of the proteomic changes, observed in both the wild-type and mutant, could have been predicted from the transcript data. The response of 5 additional proteins could be predicted from transcript data for the wild-type and another 4 protein responses could be predicted in the mutant. The protein response for 5 ORFs could not be predicted from the significant transcript data. These unpredicted responses included GroES, GroEL, rehydrin, CpcC1, and Gap2. Perhaps the most significant are GroEL (spots 278, 279 and 286) and Gap2 (spots 551 and 554), which are represented by more than one spot on 2D-gels that could reflect PTMs. Overall, only 23.5% (12 out of 51) of the changes in the proteomic data could be predicted from transcriptomic data. Pair-wise analysis of quantitative expression data revealed only modest overall correlation between transcript and protein responses (r = 0.54
0.70). Clearly, a substantial portion of the proteomic cold acclimation response is not captured by transcriptomic studies. Putative Model of the Cold Acclimation Response of Synechocystis

A summary of the proteins responding during cold acclimation of the wild-type and mutant are presented in Figure 2. It is immediately obvious that the majority of the data are of the highest confidence and it should be noted that confirmation of the changes was obtained from the Cy5 copy of the experiment. Only a few proteins are unique to either strain, which points to these proteins as being important in the cold acclimation response. These proteins are likely to have a substantial impact on cellular information processing and metabolism. Proteins form part of a highly connected network of components and as such can have metabolic consequences beyond their immediate molecular activity. Consequently, we have taken a network view of cellular information processing and metabolism to provide a systems-oriented interpretation of our proteomic data integrated with knowledge derived from the scientific literature. In Figure 3, we provide a putative graphical model depicting relevant aspects of cyanobacterial molecular biology 3685

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Journal of Proteome Research

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Figure 3. Model of cold-acclimation in Synechocystis. Proteomic data are overlaid onto a simplified graphical model of cellular information processing and metabolism. The model also incorporates information from the scientific literature. Cold causes lipid bilayers to adopt a gel state which is sensed by Hik33. A transcriptional response is initiated after signal transduction from Hik33 at at least one other sensor. Proteins are translated from the resulting transcripts and contribute to further metabolism. The absence of CrhR in the mutant results in altered RNA stability and translation, requiring greater flux through RNA degradation and protein folding pathways and compensation by increased ribosome biogenesis. Differential protein expression results in altered metabolism. The metabolism of the mutant is deficient in its ability to generate amino and fatty acids required for protein synthesis and remodelling of lipid bilayers respectively. Greater detail is provided in the main text. Arrows represent state transitions or metabolite fluxes. Color is used to indicate differences between the wild-type and mutant cold-acclimation responses. Green protein names indicate proteins that respond only in the wild-type. Red protein names respond uniquely in the mutant. Green and red arrows indicate fluxes or transitions that are increased in wild-type and mutant respectively.

and show both the protein changes that occur and their likely consequences on metabolic flux through the system. Details of the events represented in this model are described below. Low temperature is known to lead to a state transition of membrane lipids from a liquid crystalline (fluid) to a gel (rigid) state. This is believed to be sensed by the histidine kinase Hik33, resulting in signal transduction and the instigation of a transcriptional response. Not all cold-regulated genes are under the control of Hik33, so additional, thus far uncharacterised, signals are also involved and integrated into transcriptional reprogramming. Low temperature also results in an increase in secondary structure of mRNA as well as disrupting enzymatic processes including translation by the ribosomes. In the wild-type, CrhR is capable of reducing the level of secondary structure of mRNAs and, in a similar manner, could also prevent ribosome stalling during translation. The mutant is unable to do so. In this situation, mRNA with excessive secondary structure is degraded, a process involving Pnp, with the resultant ribonucleotides being made available for further transcription.

Translation proceeds as normal in the wild-type, with elevated expression of elongation factors G and TS compensating for reduced activity at low temperature. These factors are relatively less highly expressed in the mutant, but the additional induction of the ribosomal structural component Rps1 and the methylthiotransferase RimO suggest that some level of compensation can be achieved by increased ribosome biogenesis and enhancing the accuracy of translation. Nascent polypeptides are then appropriately folded into native states and participate in cellular metabolism. The difference in the abundance of GrpE between the wild-type and mutant suggests that the mutant requires a higher level of GrpE under cold conditions in order to maintain proteins in their native state. This is represented in the model as an increased flow through the protein folding process. Energy in the form of ATP and reducing equivalents (NADPH) are produced by photosynthesis. Decreased abundance of PsbO and phycobillisome components indicates possible differences in photosynthetic capacity brought about by decreased temperature. The decrease in abundance of PetH 3686

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Journal of Proteome Research (FNR) suggests that the mutant strain is likely to be further impaired in photosynthetic capacity. The products of photosynthetic electron transport are then used to fix CO2 which enters further metabolism in the form of glycerate-3-phosphate. In the wild-type, additional carbon is supplied by the breakdown of glycogen mediated in part by the induction of MalQ. Carbon sources are then delivered to both the pentose phosphate pathway and the TCA cycle. These pathways are responsible for the provision of the remaining metabolites required for cold-acclimation, including nucleotides, amino acids and, importantly, fatty acids. Incorporation of de novo synthesized fatty acids into membrane lipids and the action of acyllipid desaturases results in the alteration of membrane physical state from gel to liquid crystalline and terminates the Hik33-mediated transcriptional response. In the mutant, MalQ is not induced, restricting carbon availability to downstream metabolic processes. In addition, Pgm is down-regulated and restricts the flow of carbon into the TCA cycle. This severely compromises the mutant’s ability to synthesize fatty acids and also has a negative effect on amino acid synthesis. This prevents the fluidization of the membrane and further limits the capacity for protein synthesis, resulting in a diminished capacity for cold-acclimation.

’ CONCLUSIONS To the best of our knowledge, it is currently not possible to predict either of the molecular targets (transcripts/proteins) that can be regulated by CrhR. This information is important for a complete understanding of the cold acclimation response in Synechocystis and is potentially present in our proteomic data. Using data on the proteomic response of the wild-type and ΔcrhR strains, in combination with the transcriptomic data, it should be possible to determine whether CrhR exerts a regulatory influence at the level of translation/stability of specific proteins. If an effect exists for any specific protein, it would be manifested in one of two ways: Either (1) a transcriptional response that is the same in both strains but giving a different protein response or (2) a transcriptional response that is different but results in the same protein response. Where such proteins show a greater magnitude of change in the mutant, this would indicate a negative translational regulatory effect. Proteins that are potentially regulated at a translational/protein stability level by CrhR are annotated as such in Supplementary Table 2 (Supporting Information). It is possible that the responses of these candidates are the result of secondary and/or tertiary effects downstream from events directly regulated by CrhR. We have clearly defined the major protein changes that occur during cold acclimation and identified proteins that respond differently between the wild-type and ΔcrhR mutant strains. We have linked this to potential metabolic consequences and have strong suggestions for the role of three hypothetical proteins in the cold acclimation process. It is clear from the data and the model proposed above that the absence of CrhR has a dramatic effect on overall cellular metabolism. This is probably due to the fact that CrhR is located very early on in the response pathway. Although it is located at the interface between transcription and translation, we have shown that mutation of crhR does not result in a general dysregulation of protein synthesis. Specific differential protein responses are observed, particularly in the PPIase, chaperonin and ribosomal classes of proteins. Deficiencies in protein folding lead to further effects on specific metabolic enzymes, in addition

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to those that are possibly directly regulated by CrhR. This results in severe metabolic deficiencies that prevent the mutant from mitigating the stress.

’ ASSOCIATED CONTENT

bS

Supporting Information Supporting Information Available:Supplementary Table 1: Protein identifications and summary statistics. Supplementary Table 2: Comparison of proteomic and transcriptomic data. Supplementary Figure S1: Gross proteomic profiling of the Wildtype cold acclimation response. Supplementary Figure S2: Cold acclimation responses and differences between wild-type and ΔcrhR strains. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Antoni R. Slabas, School of Biological & Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, United Kingdom. E-mail: [email protected]. Tel: 0191 3341354. Fax: 0191 3341295.

’ ACKNOWLEDGMENT We acknowledge the support provided by the North East Proteome Analysis Facility (NEPAF), UK. ’ ABBREVIATIONS: 2D-DIGE, 2-dimensional difference in-gel electrophoresis; A730, absorbance at 730 nm; ACP, acyl carrier protein; ANOVA, analysis of variance; ATP, adenosine triphosphate; BLAST, basic local alignment search tool; CID, collision induced dissociation; DNA, deoxyribonucleic acid; FDR, false discovery rate; FNR, Ferredoxin NADP+ Reductase; GTP, guanine triphosphate; KEGG, kyoto encyclopaedia of genes and genomes; MALDI, Matrix-assisted laser desorption ionization; mRNA, messenger RNA; MS, mass spectrometry; NCBI, National Centre for Biotechnology Information; OD, optical density; ORFs, open reading frames; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PPIases, peptidyl-prolyl cis-trans isomerases; PS, photosystem; qRT-PCR, quantitative reverse transcriptase-coupled polymerase chain reaction; RNA, ribonucleic acid; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA, tricarboxylic acid cycle; TOF, time of flight ’ REFERENCES (1) Rowland, J. G.; Pang, X.; Suzuki, I.; Murata, N.; Simon, W. J.; Slabas, A. R. Identification of Components Associated with Thermal Acclimation of Photosystem II in Synechocystis sp PCC6803. PLoS One 2010, 5(5): e10511. (2) Phadtare, S.; Inouye, M. Genome-wide transcriptional analysis of the cold shock response in wild-type and cold-sensitive, quadruple-cspdeletion strains of Escherichia coli. J. Bacteriol. 2004, 186 (20), 7007– 7014. (3) Fowler, S.; Thomashow, M. F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 2002, 14 (8), 1675–1690. (4) Hannah, M. A.; Heyer, A. G.; Hincha, D. K. A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genet. 2005, 1 (2), 179–196. 3687

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