Comparative Temporal Proteomics of a Response Regulator

Comparative Temporal Proteomics of a Response Regulator (SO2426)-Deficient Strain and Wild-Type Shewanella oneidensis MR-1 During Chromate Transformation Karuna Chourey,†,¶,# Melissa R. Thompson,‡,§,3,# Manesh Shah,| Bing Zhang,⊥ Nathan C. VerBerkmoes,§ Dorothea K. Thompson,*,O and Robert L. Hettich*,§ Environmental Sciences Division, Graduate School of Genome Science and Technology, ORNL-UTK, Chemical Sciences Division, Biosciences Division, Oak Ridge National Laboratory, Tennessee 37831, Department of Biomedical Informatics, Vanderbilt University, Nashville, Tennessee 37232, and Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received September 14, 2008

Predicted orphan response regulators encoded in the Shewanella oneidensis MR-1 genome are poorly understood from a cellular function perspective. Our previous transcriptomic and proteomic analyses demonstrated that an annotated DNA-binding response regulator, SO2426, was significantly up-regulated in wild-type S. oneidensis cells at both the mRNA and protein levels in response to acute chromate [Cr(VI)] challenge, suggesting a potential regulatory role for this protein in metal stress pathways. To investigate the impact of SO2426 activity on chromate stress response at a genome-wide scale, we describe here comparative and temporal proteome characterizations using multidimensional HPLC-MS/MS and statistical analysis to identify differentially expressed proteins in biological replicates of wild-type S. oneidensis MR-1 and a so2426 deletion (∆so2426) strain, which exhibited an impaired Cr(VI) transformation rate compared to that of the parental strain. Global protein profiles were examined at different time intervals (0, 1, 3, 4 h) following exogenous chromate challenge. Results indicated that deletion of the so2426 gene negatively affected expression of a small protein subset (27 proteins) including those with annotated functions in siderophore biosynthesis (SO3032), Fe uptake (SO4743), intracellular Fe storage (Bfr1), and other transport processes. Cr(VI) exposure and subsequent transformation dramatically increased the number of differentially expressed proteins detected, with up-regulated abundance patterns observed largely for proteins involved in general stress protection and detoxification strategies, cell motility, and protein fate. In addition, the proteome data sets were mined for amino acids with potential post-translational modifications (PTMs) indicative of a level of gene expression regulation extending beyond the transcriptional control imposed by SO2426. Keywords: Shewanella oneidensis • response regulator-deficient strain • chromate challenge • liquidchromatography-mass spectrometry • linear trapping quadrupole • shotgun proteomics • posttranslational modifications

Introduction Shotgun proteomics can be used to probe gene function as well as alterations in global protein machinery as a result of targeted gene knockouts, thus, leading to increased under* To whom correspondence should be addressed. Senior author for correspondence on mass spectrometry and proteomics: R. L. Hettich, Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008 MS-6131, Oak Ridge, TN 37831-6131. Phone: (865) 574-4968. Fax: (865) 576-8559. E-mail: [email protected]. Senior author for correspondence on Shewanella biology and SO2426: D. K. Thompson, Department of Biological Sciences, Purdue University, 915 West State St., West Lafayette, IN 47907-2054. Phone: (765) 496-8301. Fax: (765) 494-0876. E-mail: [email protected]. † Environmental Sciences Division, Oak Ridge National Laboratory. ¶ Present address: Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831. ‡ Graduate School of Genome Science and Technology, ORNL-UTK. § Chemical Sciences Division, Oak Ridge National Laboratory. # These authors contributed equally to this manuscript. 3 Present address: Pharmaceutical Sciences, Pfizer, Inc., St. Louis, MO 63141. | Biosciences Division, Oak Ridge National Laboratory. ⊥ Vanderbilt University. O Purdue University. 10.1021/pr800776d CCC: $40.75

 2009 American Chemical Society

standing of cellular molecular networks responsive to environmental perturbations. Such investigations require the analysis of biological replicates and measurements of dynamic protein abundance. Most shotgun proteomics studies to date, however, have analyzed less biologically meaningful technical replicates1-5 and have provided static measurements of protein abundance. Technical replicates profile the variation between multiple LCMS (Liquid chromatography-Mass spectrometry) runs on a single biological sample,6 whereas biological replicates examine the inherent variation between samples grown under similar conditions at the same time. Recently, there have been a number of shotgun proteomics studies7-9 and a review article6 emphasizing both the informational value and necessity of incorporating biological replicates when quantifying measured proteins. Much of the discussion on the use of technical versus biological replicates has focused on the statistical treatment of the data sets.6,9-11 The primary argument for including biological replicates is to reduce any inherent background biological noise in cellular samples due to factors such as Journal of Proteome Research 2009, 8, 59–71 59 Published on Web 01/02/2009

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nutrient availability and temperature fluctuations, thereby taking into account the heterogeneity of batch cell cultures in particular. Label-free quantitative proteomics has become a popular approach for proteome quantification due to simplicity, relatively low cost, and the fact they can be used on any sample assuming proper experimental design is implemented. These approaches generally employ intrinsic MS measurement values such as peak intensities/areas of peptides,12 spectral counts,3 or normalized spectral abundance factors7 to quantify peptides and thus proteins. With the expansion of these techniques in recent years, it has become clear that statistical methods are needed to handle the large data sets and validate the results.13,10,14,15 In the study described here, the cellular role of the Shewanella oneidensis MR-1 response regulator, SO2426, is explored further by comparing dynamic proteomic profiles of a SO2426 knockout strain with wild-type S. oneidensis during chromate challenge and transformation. Species belonging to the γ-proteobacterial genus Shewanella are Gram-negative facultative anaerobes, some of which have been shown to demonstrate both direct enzymatic16–18 and indirect chemical19–21 reduction of hexavalent chromium [chromate or Cr(VI)], a widespread anthropogenic contaminant due to its use in numerous industrial and defense applications.22 The reduction of Cr(VI) to sparingly soluble Cr(III) hydroxides via the metabolic activities of microbes is a promising low-cost alternative to conventional physical-chemical treatment methods.23 We have previously described the S. oneidensis MR-1 proteome in response to acute Cr(VI) challenge,1 a dose range of Cr(VI),24 and chronic Cr(VI) exposure25 based on technical replicate measurements. Our previous work with Cr(VI)-challenged S. oneidensis MR-1 cells identified the expression of over 2400 proteins encoded in the MR-1 genome,1,24,25 with a subset of proteins demonstrating differential expression in response to Cr(VI) exposure. A surprisingly large number of these differentially expressed proteins had predicted functions in metal ion transport/binding and sulfur transport/metabolism. In addition, a functionally uncharacterized DNA-binding response regulator, SO2426, was also identified as being up-regulated at both the mRNA and protein levels in response to acute Cr(VI) exposure.1,24 Response regulators are part of two-component signal transduction systems that serve as a basic stimulus-response coupling mechanism, allowing prokaryotes to convert environmental signals to specific adaptive responses.26 In another study, the so2426 gene was also found to be induced after exposure to acute nonradioactive strontium stress.27 These observations suggest that SO2426 may be a response regulator for transition metal redox state. To examine the biological effect of a so2426 gene deletion, proteomics followed by statistical analyses was employed to measure global and temporal protein profiles in biological replicates of two different S. oneidensis MR-1 strains, the wild-type and an SO2426-deficient mutant. Differential regulation of protein function through the identification of post-translational modification (PTM) sites on proteins present in the global data set was also investigated.

Experimental Methods Reagents and Sample Preparation. Luria-Bertani (LB) medium (BD Diagnostic Systems, MD), guanidine HCl, Tris, EDTA, ammonium acetate, and CaCl2 were purchased from Sigma Chemical Co. (St. Louis, MO) and were used without 60

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further purification. Modified sequencing grade trypsin was purchased from Promega (Madison, WI) and used for all protein digestions. The 99% formic acid was obtained from EM Science (Darmstadt, Germany) and HPLC-grade water and acetonitrile (ACN) from Burdick & Jackson (Muskegon, MI). Three independent cultures (or biological replicates) each of wild-type S. oneidensis MR-1 and strain ∆so242628 were grown aerobically in parallel in 250 mL flasks with 100 mL of LB medium (pH 7.2) at 30 °C with constant agitation at 200 rpm. When the OD at 600 nm reached 0.5 (midlog phase), a 10-mL aliquot of each culture was taken as the 0 time point and processed as described in Brown et al.1 Cr(VI) in the form of K2CrO4 was then added to all six cultures at a final concentration of 0.3 mM. Extracellular Cr(VI) concentration in the media was monitored using the 1,5-diphenylcarbazide (DPC) method as described previously.29 When the approximate Cr(VI) concentration for the wild-type S. oneidensis cultures reached 0.25 (1 h), 0.15 (3 h), and 0 (4 h) mM, 10 mL from all six cultures (wild-type and mutant) was harvested for LC-MS/MS analysis. Cell Lysis and Digestion. The resulting frozen cell pellets from the 10 mL aliquots were weighed and then further distributed into aliquots of 5 mg wet cell pellet for lysis. The lysis protocol used was essentially as described in Thompson et al.30 Briefly, 6 M guanidine with 10 mM DTT were added to each sample and incubated at 37 °C overnight after which the guanidine was diluted 6-fold with 50 mM Tris/10 mM CaCl2. Then 5 µg of trypsin was added, followed by a 6 h incubation period after which another aliquot of trypsin was added with an overnight incubation at 37 °C. A final reduction step for 2 h was performed with 20 mM DTT. Protein digests were then spun at 10 000g for 10 min to remove cellular debris and then stored at -80 °C until the LC/LC-MS/MS experiment. Global Proteome LC/LC-MS/MS Analysis. The LC/LC-MS/ MS experiments were performed as described in Brown et al.1 Each lysis/digestion from each culture was a separate 24 h LC/ LC-MS/MS experiment. Samples were loaded onto a split phase column consisting of reverse phase (C18) and strong cation exchange (SCX) separation materials. This column was placed behind a 15 cm C18 analytical column located directly in front of the mass spectrometer (LTQ, Thermo Scientific, San Jose, CA). The LTQ was coupled with an Ultimate HPLC pump (LC Packings, a division of Dionex, San Francisco, CA). The samples were analyzed with a 12-step 2D HPLC analysis by adding increasing concentrations (0-500 mM) of ammonium acetate salt pulses followed by an aqueous (95% H2O, 5% ACN, 0.1% formic acid) to organic (30% H2O, 70% ACN, 0.1% formic acid) gradient. The LTQ was operated in the data-dependent mode during the chromatographic separations as detailed in Brown et al.1 Proteome Bioinformatics and Data Analysis. The protein database used for all MS searches consisted of Version 8 (www.tigr.org) of the S. oneidensis MR-1 proteome (4798 proteins) as well as 36 common contaminant sequences (trypsin, keratin, etc.). The database is available for download at the project Web site http://compbio.ornl.gov/shewanella_metal_stress/databases/. Initially, Sequest31 was used to search the resulting MS raw files for peptide/protein identifications. Sequest searches were performed as detailed in Brown et al.1 with the following scoring cutoffs. A minimum of two peptides was required for a positive identification of a protein with peptide charge state score minimums of 1.8 (+1), 2.5 (+2), and 3.5 (+3). A deltCN value of 0.08 was required for peptide

Comparative Temporal Proteomics of SO2426-Deficient Strain identifications. Sequest results were then filtered and sorted according to the above criteria with DTASelect and Contrast,32 and all resulting data are available on the project Web site (http://compbio.ornl.gov/shewanella_metal_stress/reduction). The resultant proteome data sets were analyzed using the Poisson regression model.33 The Poisson regression model is commonly used for count data. It assumes the count data has a Poisson distribution, a distribution that we frequently encounter when we are counting a number of events, and assumes the logarithm of its expected value can be modeled by a linear combination of the independent variables. In our context, the spectral counts for a protein in different experiments were the count data. As we evaluated genotype and treatment separately, we only had a single independent variable in each model (genotype or treatment). To make the spectral counts comparable across different experiments, we normalized the spectral counts for a protein to the total spectral counts. In Poisson regression, this is handled by adding the logarithm of total spectral count as an independent variable with a fixed coefficient of 1. The p values generated by the model were further adjusted using the Benjamini and Hochberg correction to account for multiple comparisons.34 A False Discovery Rate (FDR) of 1% was used to select for proteins that were differentially expressed between the two groups under comparison. We compared the ∆so2426 and wild-type proteomes under conditions of no added chromate and at 1, 3, and 4 h following chromate amendments to the growth medium. PTM Mining. For performing searches for post-translational modifications (PTMs), the search algorithm InsPecT35 was used. For optimization of PTM searches with InsPecT on shotgun proteomics data, see ref 36. The proteome database used in these searches consisted of the forward database (mentioned above) with the reversed sequences of all proteins concatenated to the end. This database was used in order to estimate the false discovery rate (FDR)37 of each individual search. MS/MS spectra were extracted from the raw files using ReAdW.exe [Institute for Systems Biology in Seattle, WA (http://www.systemsbiology.org)] to create an mzXML file. The mzXML files were then searched with InsPecT35 specifying the following PTMs: An optional mass of 14 Da (monomethylation) was added to lysines, arginines, and glutamates; optional masses of 28 (dimethylation), 42 (trimethylation/monoacetylation), and 84 (diacetylation) Da were added to lysines and arginines; 16 Da (monooxidation) to methionines, cysteines, tyrosines, and tryptophans; 32 Da (dioxidation) to methionines and cysteines; and 48 Da (trioxidation) on cysteines. The scripts Pvalue.py and Summary.py (components of the InsPecT software package) were used to filter the resulting output tab-delimited text files by p-value and peptide count. A peptide count of two was required for all proteins, and the p-value cutoff was adjusted to give a false discovery rate of 2 ( 0.2% for each unmodified proteome data set.

Results and Discussion Comparison of Chromate Transformation Rates. The S. oneidensis MR-1 so2426 gene encodes a putative DNA-binding response regulator that was implicated in our previous global studies1,24 in the regulation of the cellular response to acute chromate challenge. SO2426 contains an N-terminal CheY-like domain and a C-terminal winged-helix DNA-binding domain, which is annotated as a member of the Trans_reg_C family (PF00486) based on the Pfam database.38 SO2426 is highly conserved at the amino acid level to homologues from 15 other

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Figure 1. Cr(VI) disappearance from the medium is depicted for S. oneidensis cultures of wild-type MR-1 (square symbols) and the ∆so2426 mutant (triangle symbols). No detectable Cr(VI) reduction was observed for the abiotic LB-only control (black diamonds).

Shewanella genomes sequenced to date.24 In a previous study, the cre-lox recombination method was used to create a nonpolar in-frame deletion of the so2426 locus in order to assess the impact of SO2426 on the transcriptomics of the S. oneidensis response to heavy metal stress.28 Wild-type S. oneidensis MR-1 and the ∆so2426 mutant were cultivated aerobically in LB medium until the cultures reached midlog phase (OD600, 0.5) and then the cells were challenged with 0.3 mM K2CrO4 (see Experimental Methods). Chromate disappearance from the culture medium was monitored using the DPC assay at 1, 3, and 4 h following chromate addition (Figure 1). The experiment was terminated at the 4 h time interval, because wild-type cells had completely transformed the chromate in the medium to nondetectable levels. By contrast, the ∆so2426 mutant had transformed approximately 57% of the extracellular Cr(VI) at the 4 h time point, indicating that the SO2426-deficient strain was impaired in its ability to reduce chromate (Figure 1). Cell samples for both the wildtype and mutant were harvested at different time points during the chromate transformation profile and subjected to LC-MS/ MS analysis to understand how their proteomes changed temporally during Cr(VI) challenge and in relation to the so2426 deletion. The slower extracellular Cr(VI) removal rate exhibited by the mutant indicates that, although the so2426 gene is not required for chromate transformation, it plays a possible role in the overall fitness of the bacterium to cope effectively with chromate stress. Global Protein Profiles of Wild-Type S. oneidensis MR-1 and the ∆so2426 Mutant. Label-free quantitation was used to determine whether proteins were differentially expressed between the two strains, and was evaluated with a detailed statistical analysis of spectral counts. Traditionally, label-free quantitation is applied to data sets derived from technical replicates analyzed using HPLC-MS/MS; here, we utilize biological replicates instead of technical replicates, with the understanding that more inherent variation will be observed in the biological replicates. However, the average Pearson’s Journal of Proteome Research • Vol. 8, No. 1, 2009 61

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Table 1. Total Number of Nonredundant Proteins Detected in Strains MR-1 and ∆so2426 at Each Time Point Time (h) post-Cr(VI) addition

Amount (mM) of Cr(VI) remaining (WT/mutant)

Wild-type MR-1a

∆so2426 mutanta

0 1 3 4

∼0.3/0.3 ∼0.3/0.3 ∼0.15/0.18 0/0.13

1537 1583 1535 1468

1398 1540 1599 1687

a Total number of nonredundant proteins identified based on three biological replicates.

correlation coefficient for pairs of biological replicates in this study was found to be 0.95. In total, 2121 proteins (44% of the predicted proteome) were confidently identified at the two-peptide level for strains MR-1 and ∆so2426. Table 1 depicts the total number of proteins identified for strains MR-1 and ∆so2426 at the different time intervals during chromate challenge and the corresponding reductions in extracellular chromate reduction. The molecular weight and isoelectric point distribution for the data sets was plotted and demonstrated no discrepancy when compared to the predicted distribution of the proteome (data not shown). The proteins detected for each strain were grouped according to their role category assignments as provided by the J. Craig Venter Institute (formerly, The Institute for Genomic Research), and the distribution results are presented in Figure 2. Generally, the numbers of proteins belonging to the different functional classes were similar across all the time intervals examined. This was also true for the protein expression profiles between the two strains. However, fluctuations were noted within individual role categories over the time course of the experiment. Differences between the ∆so2426 and Wild-Type Proteomes under Conditions of No Exogenous Chromate. To directly assess the effect deletion of the SO2426 response regulator gene had on global protein profiles, the differential proteomes of exponentially growing ∆so2426 and wild-type cells were characterized prior to the addition of exogenous chromate (i.e., the 0 h time point). A relatively small subset of 42 proteins was measured as being differentially expressed at significant levels (FDR < 0.01) between the two different S. oneidensis strains at the 0 h time point, with the majority corresponding to down-regulated proteins (Table 2). A complete protein profile of wild-type and mutant cells under conditions of no chromate is presented in Supplemental Table S1 in Supporting Information. Most notably, proteins with predicted cellular functions in siderophore-mediated Fe binding and transport (SO3032, SO4743), intracellular Fe storage (Bfr1), or those shown previously to be associated with Fe homeostasis (SO1190)39 were down-regulated in the mutant relative to the wild-type. Furthermore, the expression patterns for these proteins remained consistently down-regulated throughout the time course of chromate exposure. A previous transcriptomics analysis of the ∆so2426 mutant demonstrated that, with the exception of SO1112 (Bfr1), the mRNA levels for SO1190, SO3032, and SO4743 showed a corresponding reduction under chromate conditions, suggesting that these iron-dependent proteins may be potential direct targets of positive transcriptional control by SO2426 in S. oneidensis.24 Interestingly, the same study showed that another iron storage gene, ftn (so0139) encoding ferritin, was strongly down-regulated at the transcription level in the ∆so2426 mutant under Cr(VI) challenge.24 This is 62


consistent with results obtained in this study, in which the protein ferritin was measured as being slightly down-regulated in the mutant in response to chromate exposure (Supplemental Tables 2, 3, and 4 in Supporting Information). SO1405 (transglutaminase family protein), SO1821 (putative outer membrane porin), two molybdenum ABC transporters (ModA, SO3967), and several other proteins of unknown function (SO3077, SO4403, SO4509) also were detected at decreased abundance levels in the mutant regardless of whether chromate was present, indicating that differential expression of these proteins might be a consequence of the so2426 gene deletion, although this is speculative without further research. Collectively, the proteomic data acquired at the 0 h time interval suggest a functional role for SO2426 that extends beyond the initial hypothesis of SO2426 as a regulator involved in chromate stress response and includes a connection with the regulation of Fe homeostasis. Proteins encoded by genes arranged in the probable operon so4505-so4513 (formate dehydrogenase) were also downregulated to varying degrees in the deletion mutant in the absence (Table 2) or presence of chromate (Tables 3-5). Formate dehydrogenases constitute a complex of enzymes that catalyze the oxidation of formate by donating the electrons to a second substrate, such as NAD+ or a cytochrome.40 These enzymes are known to depend on Fe, Se, Mo, and tungsten for optimal activity.41 In addition, SO3669, annotated as the heme transport protein HugA, showed slightly higher abundance levels in the mutant compared to the wild-type at the 0 h time point (no chromate) and in the presence of Cr(VI) (Supplemental Table S5 in Supporting Information). This protein is predicted to function in iron transport, and the upregulated expression profile of HugA may indicate perturbation of intracellular iron pools in the so2426 deletion mutant. Similarly, repression of the nonessential iron-containing formate dehydrogenase complex may be a direct consequence of intracellular iron deficiencies in the mutant, although this is purely speculative at this time. Temporal Changes in the ∆so2426 Proteome during Chromate Challenge and Reduction. In general, exposure of both the wild-type and mutant strains to 0.3 mM chromate resulted in a substantially larger number of differentially expressed proteins identified at each post-Cr(VI) time interval relative to the 0 h time point: 1 h (106 proteins), 3 h (69 proteins), and 4 h (146 proteins) (Supplemental Tables 2-4 in Supporting Information). The most highly up- and downregulated proteins detected in the different chromate-perturbed temporal proteomes are presented in Tables 3-5. Overall, various molecular chaperones, antioxidants, and proteins involved in general stress protection or detoxification mechanisms were identified as being up-regulated in the mutant at each of the three time points during Cr(VI) exposure (Tables 3-5, Supplemental Tables 2-4 in Supporting Information) and included the following: glyoxalase family proteins SO3586 (1 h) and SO1756 (3 h); universal stress protein SO3681 (1 h); proteases ClpB and HslV (1 h); DnaK (3 and 4 h); GroES (3 h); alkyl hydroperoxide reductase, subunits C (AhpC) and F (AhpF) (4 h); and glutathione synthetase GshB (4 h). These results suggest that chromate-induced oxidative stress in the mutant might be more pronounced, presumably due to the so2426 gene deletion and the resulting reduced capacity of the mutant to properly regulate intracellular Fe pools. Other notable temporal expression patterns characterizing the mutant were an increase in the synthesis of proteins involved in powering (i.e., generat-

Comparative Temporal Proteomics of SO2426-Deficient Strain

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Figure 2. Functional category distribution for proteins detected from S. oneidensis strains MR-1 and ∆so2426. Functional category distribution for each strain (A) prior to Cr(VI) exposure, (B) 1 h post-Cr(VI) exposure, (C) 3 h post-Cr(VI) exposure, and (D) 4 h postCr(VI) exposure. The numbers along the x-axis refer to the following functional role categories: (1) amino acid biosynthesis; (2) biosynthesis of cofactors, prosthetic groups, and carriers; (3) cell envelope; (4) cellular processes; (5) central intermediary metabolism; (6) DNA metabolism; (7) energy metabolism; (8) fatty acid and phospholipid metabolism; (9) hypothetical proteins; (10) mobile and extrachromosomal element functions; (11) protein fate; (12) protein synthesis; (13) purines, pyrimidines, nucleosides, and nucleotides; (14) regulatory functions; (15) signal transduction; (16) transcription; (17) transport and binding proteins; and (18) unknown function.

ing energy) and controlling flagellar rotation (FliI, FliN, CheR) at the 3 and 4 h time points, but a decrease in the abundance levels of various flagellar structural proteins comprising the hook (FlgE), P ring (FlgI), and basal-body rod (FlgG) (Tables 4 and 5; Supplemental Tables 3 and 4 in Supporting Information). In addition to the transport and binding proteins downregulated at the 0 h time point, the chromate-perturbed proteome of the mutant was characterized by a decrease in the abundance levels of FeoB (1 h), the iron-storage protein Ftn (1, 3, and 4 h), the siderophore biosynthesis protein AlcA (1 and 3 h), and a ferric alcaligin siderophore receptor (1, 3, and 4 h) (Tables 3-5, Supplemental Tables 2-4 in Supporting Information). FeoB, a G protein-like transporter different from siderophore-dependent systems, is involved in ferrous iron [Fe(II)] uptake,42 and AlcA is encoded by the first gene in a siderophore biosynthesis operon comprising so3030, so3031, and so3032. Siderophores are low-molecular-weight ferric iron [Fe(III)] chelators that are synthesized and secreted by bacterial cells to scavenge iron from the surrounding milieu under conditions of environmental iron deficiency.42 Presumably, SO3033 is the outer membrane receptor that specifically

recognizes the siderophore synthesized by the enzymes encoded in the alcA-so3031-so3032 operon. This connection between iron transport and chromate stress response has been observed in our previous work,1,24 suggesting that the intracellular balance of Fe(II) pools may be disrupted by exposure to chromate, although this has yet to be empirically validated. FeoB, AlcA, and SO3033 were shown previously to be upregulated in chromate-treated wild-type S. oneidensis cells in contrast to untreated control cells1 but had decreased abundance in the ∆so2426 strain under chromate conditions. Collectively, these data suggest that the primary physiological function of the S. oneidensis SO2426 response regulator may be in siderophore-mediated iron transport and homeostasis. By contrast, the heme transport protein HugA and IrgA (ironregulated outer membrane virulence protein) showed low levels of increased synthesis in the mutant across all four time points (Supplemental Tables 1-4 in Supporting Information). It was previously suggested that S. oneidensis IrgA is likely involved in iron acquisition based on the observation that IrgA from Vibrio cholerae is most closely related to iron-regulated ferric siderophore receptors.43 The increased abundance of iron Journal of Proteome Research • Vol. 8, No. 1, 2009 63

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Table 2. Differentially Expressed Proteins Detected at the 0 h Time Interval (No Chromate) Gene ID

Mutant/WT ratio

p-value

SO0444 SO2652

23.53 24.43

0.000168 0.0003573

SO2757

19.81

9.36 × 10-13

SO4513 SO4510 SO1863 SO3032 SO3863

-1.58 -2.01 -2.11 -2.17 -2.22

7.00 × 10-9 4.01 × 10-5 0.0002928 0.0001595 1.89 × 10-23

SO3967

-2.89

1.03 × 10-9

SO4403 SO1112 SO0105 SO0809 SO4509 SO3077 SO1821 SO1190 SO1405 SO4743 SO0348

-20.48 -20.57 -21.48 -21.48 -22.44 -22.48 -22.74 -22.84 -23.01 -23.61 -23.88

0.0002297 0.0001117 0.0002297 0.0002297 9.61 × 10-23 0.0002297 2.67 × 10-5 3.46 × 10-13 1.56 × 10-6 8.81 × 10-11 6.43 × 10-6

Gene

Gene product

Up-Regulated Proteins Hypothetical proteins prophage MuSo2, transcriptional regulator, Cro/CI family membrane protein, putative Down-Regulated Proteinsa formate dehydrogenase, alpha subunit fdhB-1 formate dehydrogenase, alpha subunit DNA-binding protein, HU family siderophore biosynthesis protein, putative modA molybdenum ABC transporter, periplasmic molybdenum-binding protein molybdenum ABC transporter, periplasmic molybdenum-binding protein, putative hypothetical protein bfr1 bacterioferritin subunit 1 selA L-seryl-tRNA selenium transferase azu azurin precursor hypothetical protein conserved hypothetical protein outer membrane porin, putative conserved hypothetical protein transglutaminase family protein TonB-dependent receptor, putative acyltransferase family protein

Functional category

Hypothetical proteins Other categories Cell envelope Energy metabolism Energy metabolism DNA metabolism Transport and binding proteins Transport and binding proteins Transport and binding proteins hypothetical protein Transport and binding proteins Protein synthesis Energy metabolism hypothetical protein Hypothetical proteins Transport and binding proteins Hypothetical proteins Unknown function Transport and binding proteins Unknown function

a The entire subset of down-regulated proteins at the 0 h time point displayed decreased abundance profiles across the entire time course of chromate exposure.

transport proteins indicates a perturbation in iron availability that is more pronounced in the mutant compared to the wildtype. In contrast to the 1-h and 3-h Cr(VI)-perturbed proteomes, the 4-h proteome of the mutant was distinguished by a greater number of down-regulated energy metabolism proteins, particularly cytochromes and iron-sulfur proteins (PetA, OmcA, SO1521, SO3420) involved in electron transport processes. OmcA is a decaheme cytochrome involved in the reduction of extracellular insoluble Mn(IV) or Fe(III) oxides44 and was identified as being down-regulated in the mutant only at the 4 h time interval, at which point the mutant had removed approximately 58% of the external chromate, whereas wildtype MR-1 had removed 100% of the external chromate (Figure 1). Other proteins down-regulated in the mutant relative to the wild-type strain that cannot be explained at this point included a putative chitin-binding protein (SO1072; 1, 3, and 4 h), SurA (a periplasmic molecular chaperone that facilitates correct folding of outer membrane porins;45 1 h), and ModA (molybdenum ABC transporter; 1, 3, and 4 h). A comprehensive data sheet documenting the spectral counts and mutant/WT ratios at all time points is presented in Supplemental Table S5 in Supporting Information. Post-Translational Modifications of Proteins in S. oneidensis during Cr(VI) Reduction. Protein PTMs such as methylations, phosphorylations, and acetylations are known to regulate gene expression and protein-protein interactions.36 To address this goal of obtaining more extensive proteome information, the temporal proteomics data sets for wild-type S. oneidensis were mined for modified proteins, with a particular emphasis on modifications to the response regulator SO2426. The MS/MS spectra acquired for all four time points (0, 1, 3, and 4 h post chromate addition) for S. oneidensis were searched using the computational algorithm InsPecT to identify 64


PTMs not targeted for identification during the initial Sequest search. It has been shown previously that InsPecT is a suitable tool to search for characterizing multiple modifications in a large proteome data set.35,36 This search strategy depends on a scoring scheme that ultimately assigns a p-value to each peptide match, so that peptide identification with an acceptable false discovery rate (FDR), in our case set to be 2.0 ( 0.2%, can be achieved across the data set by setting a p-value cutoff. Initially, a forward database is created that contains a comprehensive list of S. oneidensis proteins and common contaminants. Then, a reverse database is generated that contains the reversed sequences for the same proteins, and this is concatenated to the forward database. InsPecT utilizes a sequence tag approach to rapidly filter the database, such that possible PTMs are considered for a relatively short list of candidate sequences. Next, match quality scores are assigned based on agreement of spectral features between the observed and theoretical tandem mass spectra for either unmodified or modified peptides, in the latter case incorporating the specified PTM mass shifts. For a correctly identified peptide, the fragmentation pattern will generate an acceptable match only with an entry in the forward database but will not match the reverse database. The p-value for the candidate peptide is determined based on comparison of the match quality score (somewhat analogous to the cross-correlation (Xcorr) score of Sequest) against the distribution of quality scores among matches to the reverse database, and a p-value cutoff is determined such that the FDR remains approximately 2%.35,36 Table 6 contains the p-value cutoff chosen for each data set with the corresponding FDRs observed. The optimization of the filtering threshold for identification of PTM peptides to a specific FDR leads to a more accurate comparison across the information in the data sets. The p-value distribution was from 0.45 to 0.83 for S. oneidensis cultures sampled at the four time

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Comparative Temporal Proteomics of SO2426-Deficient Strain Table 3. Selected Differentially Expressed Proteins Detected at the 1 h Post-Cr(VI) Time Interval Gene ID

SO2578 SO3586 SO2769 SOA0080 SO0861 SO0576 SO2236 SO0934 SO0278 SO4378 SO0582 SOA0164 SO3434 SO1652

Mutant/WT ratio

22.47 22.58 23.58 24.35 22.35 21.67 21.35 21.22 21.05 20.84 20.35 20.35 20.35 19.58

p-value

Gene

0.00029 0.00013 0.00013 0.00063 0.00063 6.13 × 10-5 0.00063 1.39 × 10-7 1.34 × 10-6 1.32 × 10-5 0.00063 0.00063 0.00063 0.00013

minE crr argG tpm pcm smpA tatA -

SO1476 SO0527 SO4202 SO0004 SO1313 SO2564 SO2967 SO2419

-19.52 -20.32 -20.42 -20.42 -20.52 -20.61 -20.62 -21.76

0.00014 0.00058 0.00029 0.00029 0.00014 7.11 × 10-5 4.47 × 10-11 1.78 × 10-5

SO1405 SO1124 SO1597 SO1404 SO2356 SO4323 SO0968 SO2426 SO1429 SO4510 SO1751 SO4513 SO4743 SO3103 SO1190 SO4509

-21.83 -22.32 -22.52 -22.52 -22.52 -22.52 -22.61 -22.76 -22.89 -23.07 -24.07 -24.21 -24.34 -24.42 -25.44 -25.97

8.94 × 10-6 0.00058 0.00014 0.00014 0.00014 0.00014 7.11 × 10-5 1.78 × 10-5 4.50 × 10-6 5.77 × 10-7 5.77 × 10-7 7.50 × 10-8 3.19 × 10-21 0.00029 3.04 × 10-23 3.79 × 10-15

Gene product

Functional category

Up-Regulated Proteins cell division topological specificity factor MinE glyoxalase family protein conserved hypothetical protein hypothetical protein conserved hypothetical protein PhoH family protein PTS system, glucose-specific IIA component conserved hypothetical protein argininosuccinate synthase FAD-binding protein thiopurine S-methyltransferase iron-containing alcohol dehydrogenase protein-L-isoaspartate O-methyltransferase conserved hypothetical protein

Cellular processes Unknown function Hypothetical proteins HP Hypothetical proteins Unknown function Transport and binding proteins Hypothetical proteins Amino acid biosynthesis Unknown function Cellular processes Unknown function Protein fate Hypothetical proteins

Down-Regulated Proteins small protein A Unknown function conserved hypothetical protein Hypothetical proteins Sec-independent protein translocase protein TatA Protein fate inner membrane protein, 60 kDa Cell envelope conserved hypothetical protein Hypothetical proteins transglycosylase, Slt family Cell envelope conserved hypothetical protein Hypothetical proteins 2,4-dienoyl-CoA reductase, putative Fatty acid and phospholipid metabolism transglutaminase family protein Unknown function conserved hypothetical protein TIGR00011 Hypothetical proteins conserved hypothetical protein Hypothetical proteins endoribonuclease L-PSP, putative Transcription etrA electron transport regulator A Regulatory functions GGDEF domain protein Unknown function ldhA D-lactate dehydrogenase Energy metabolism DNA-binding response regulator Signal transduction dmaA-1 anaerobic dimethyl sulfoxide reductase Energy metabolism formate dehydrogenase, alpha subunit Energy metabolism membrane protein, putative Cell envelope conserved hypothetical protein Hypothetical proteins TonB-dependent receptor, putative Transport and binding proteins AcrB/AcrD/AcrF family protein Cellular processes conserved hypothetical protein Hypothetical proteins hypothetical protein Hypothetical proteins

points examined. The columns representing total unmodified peptide identifications show results for the protein data set without protein modifications, with the resulting FDR remaining at 2%. The PTM Peptide Identifications column presents matches to the database where only possible protein modifications have been included. The resulting FDR for PTM-containing peptides was 12.0-19.0%, since there were many more peptide fragmentation patterns possible. The PTM identifications for monomethylations and dimethylations can be accessed in Supplemental Table S6 in Supporting Information. PTMs Determined across Multiple Data Sets. Under conditions of no added chromate, the total number of proteins expressed in the wild-type and mutant were similar (Table 1). However, proteins in the mutant showed a higher number of modified peptides. The most prevalent modifications observed in nonstressed cells (0 h time point) were oxidation of methionines, cysteines, tyrosines, and tryptophan residues, followed by diacetylation of lysines and arginines. At the 4 h time interval of chromate challenge, fewer dimethylations were identified, and there were more protein oxidations compared to the 0 h time point control. The oxidation identifications for both data sets primarily consisted of monooxidations. The

identification of a greater proportion of oxidized peptides following chromate exposure likely indicates cells experienced increased intracellular oxidative stress. Chromate reduction is known to generate the highly reactive radical Cr(V), which redox cycles, thereby producing reactive oxygen species and oxidative stress as a consequence.46,47 We have reported earlier that chromate exposure leads to up-regulation of DNA repair genes and oxidative stress protection genes/proteins in S. oneidensis.1,24,25 However, the nature of the sample preparation may also contribute to some peptide oxidation observed, especially for methionines, but is not likely to account for all the oxidation PTMs identified in the proteome data set. Even though phosphorylations are common signaling PTMs, this modification was not targeted in these searches since in bacteria, most phosphorylations occur on histidine or aspartic acid residues, which are labile and difficult to observe after sample preparation processing.48-50 In addition, the method of fragmentation utilized in this study (low energy collision induced dissociation) is often not amenable for deciphering the location of the phosphorylation site on the peptide.36 To better visualize global differences in PTM characteristics across the time course, modified peptides identified for each Journal of Proteome Research • Vol. 8, No. 1, 2009 65

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Table 4. Selected Differentially Expressed Proteins Detected at the 3 h Post-Cr(VI) Time Interval Gene ID Mutant/WT ratio

p-value

Gene

Gene product

Functional category

SO4224

23.03

1.24 × 10-6

murE

SO3255 SO0934 SO1271 SO0018 SO3220 SO3516 SO1397

25.09 22.42 22.40 22.40 22.20 21.91 21.69

6.06 × 10-7 4.10 × 10-9 0.0004188 0.0004188 1.45 × 10-7 5.25 × 10-6 4.63E-05

fliI fliN -

Up-Regulated Proteins UDP-N-acetylmuramoylalanyl-D-glutamate-2,6diaminopimelate ligase flagellum-specific ATP synthase FliI conserved hypothetical protein polyamine ABC transporter, ATP-binding protein conserved hypothetical protein flagellar motor switch protein FliN transcriptional regulator, LacI family cytosine deaminase

SO2115 SO2993 SO2441

21.50 21.20 19.91

0.00020019 1.45 × 10-7 5.25 × 10-6

-

asparaginase family protein

thiG

thiG protein

SO1551

2.44

1.01 × 10-19 -

SO4743 SO2843 SO3967

-2.00 -2.31 -3.57

2.40 × 10-12 3.44 × 10-5 7.81 × 10-20 -

Transport and binding proteins DNA metabolism Transport and binding proteins

SO3391 SO2478

-19.51 -20.40

0.00019315 0.00040544

Protein fate Cell envelope

SO3184 SO4514 SO3194 SO1030

-20.60 -21.51 -21.85 -22.51

9.24 × 10-5 0.00019315 1.03 × 10-5 0.00019315

SO1502 SO4510 SO1405 SO4513 SO4509 SO1190

-22.60 -23.25 -23.46 -23.64 -23.97 -25.01

9.24 6.59 1.84 5.26 1.07 9.33

× × × × × ×

10-5 10-8 10-9 10-11 10-14 10-39

GGDEF domain protein

Down-Regulated Proteins TonB-dependent receptor, putative exonuclease SbcC, putative molybdenum ABC transporter, periplasmic molybdenum-binding protein, putative ATP-dependent protease, putative kdsB 3-deoxy-D-manno-octulosonate cytidylyltransferase conserved hypothetical protein fdhB-2 formate dehydrogenase, iron-sulfur subunit transcriptional activator rfaH, putative metH 5-methyltetrahydrofolate-homocysteine methyltransferase cobalamin synthesis protein/P47K family protein formate dehydrogenase, alpha subunit transglutaminase family protein formate dehydrogenase, alpha subunit hypothetical protein conserved hypothetical protein

time point were organized according to the functional role category assigned to the parent protein. Figure 3 depicts the functional distribution of the peptides identified at each time point. While many proteins underwent modifications per se, the total number of modified proteins in each category did not vary substantially as a function of chromate exposure time. The protein synthesis category predominates across all time points as containing the greatest number of modified peptides. Proteins encoding ribosome components belong to this category and are known to be highly modified.51 Proteins with a role in amino acid biosynthesis demonstrated increased modifications over time, as did the proteins involved in cellular processes. The discussion outlined above describes the broad view of how protein post-translational modification changes globally as a function of chromate shock. In the following sections, two specific examples are highlighted to show how PTM information can provide a more detailed description of cellular activity than simple proteome characterization in the absence of PTM considerations. Post-Translational Modification of Chemotaxis Proteins. Reversible methylation of chemotaxis proteins is one of the principal regulatory modes for controlling microbial movement in response to environmental stimuli, thus, bringing about adaptation to the new surrounding.52-54 Bacterial movements are typically characterized by the smooth movement (or swimming) of cells favorably toward an attractant, or tumbling 66


Cell envelope Energy metabolism Hypothetical proteins Transport and binding proteins Hypothetical proteins Cellular processes Regulatory functions Purines, pyrimidines, nucleosides, and nucleotides Energy metabolism Other categories Biosynthesis of cofactors, prosthetic groups, and carriers Unknown function

Hypothetical proteins Energy metabolism Regulatory functions Amino acid biosynthesis Unknown function Energy metabolism Unknown function Energy metabolism Hypothetical proteins Hypothetical proteins

of cells away from a repellent.45,55,56 For example, CheY has been reported to be involved in the tumbling of cells,45,55 whereas CheW has been implicated in the smooth movement of cells.57 S. oneidenesis is known to have multiple chemotaxis proteins, whose complex and somewhat ill-defined interplay control bacterial movement. Inspection of the proteome data sets reveals the presence of several chemotaxis proteins; these were examined closely for modifications, in particular for methylation variation, as a function of time after chromate shock. Although the complete chemotaxis methylation story is complicated and beyond the scope of this investigation, some interesting patterns were observed from the inspection of the modified peptide data sets. In particular, methylation differences were seen in the CheW and CheY chemotaxis proteins. CheW (SO3203) was not observed at the 0 h time point, but was found as a multiple methylated version at the 1 h time point. No methylated CheW was observed at the 3 or 4 h intervals. In contrast, CheY (SO3209) was observed as heavily oxidized at all time points, but was found with multiple methylations at the 1 and 3 h time points. No methylated CheY was observed at either 0 or 4 h time points. We have observed that, when cells are exposed to hexavalent chromium, a distinct reduction in cellular motility is observed (data not shown), which typically followed the methylation time frame of CheW (Table S6 in Supporting Information). After chromate reduction is complete (∼ 3-4 h), most of the cells resume movement, in particular tumbling which again coincides with observed

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Comparative Temporal Proteomics of SO2426-Deficient Strain Table 5. Selected Differentially Expressed Proteins Detected at the 4 h Post-Cr(VI) Time Interval Gene ID

Mutant/WT ratio

p-value

Gene

Gene product

Functional category

SO3966 SO3671 SO1662 SO2047 SO1525

23.45 24.92 23.45 23.23 23.04

0.0002587 3.81 × 10-6 0.0002587 0.0010841 9.50 × 10-7

mgtE-2 exbB1 dxs

Up-Regulated Proteins magnesium transporter TonB system transport protein ExbB1 conserved hypothetical protein prolyl oligopeptidase family protein deoxyxylulose-5-phosphate synthase

SO2441

22.98

1.90 × 10-6

thiG

thiG protein

SO2986 SO0338 SO3110 SO3089

22.64 22.55 22.55 22.37

6.28 × 10-5 0.0001272 0.0001272 7.62 × 10-9

secF-2 -

hypothetical protein hypothetical protein protein-export membrane protein SecF fatty oxidation complex, beta subunit

SO1962 SO1168 SO3723 SO3388 SO1854 SO4264

22.35 22.23 22.23 22.04 21.86 21.79

0.0005283 0.0010841 0.0010841 9.50 × 10-7 7.66 × 10-6 1.54 × 10-5

mrdA cysC hsdS-2

SO4645 SO2290 SO2419

21.72 21.72 21.55

3.11 × 10-5 3.11 × 10-5 0.0001272

-

4-hydroxyphenylpyruvate dioxygenase penicillin-binding protein 2 adenylylsulfate kinase ATP-dependent RNA helicase, DEAD box family hypothetical protein type I restriction-modification system, S subunit hypothetical protein rhodanese domain protein 2,4-dienoyl-CoA reductase, putative

SO3225 SO3615 SO3392 SO4120 SO3584 SO0956 SO4403 SO2279 SO3008 SO3251 SO0861 SO4189 SO0165 SO1674 SO2228 SO2588

21.45 21.35 21.04 20.72 20.64 20.35 20.35 20.35 20.35 20.35 20.35 20.35 20.35 20.23 20.23 20.23

0.0002587 0.0005283 9.50 × 10-7 3.11 × 10-5 6.28 × 10-5 0.0005283 0.0005283 0.0005283 0.0005283 0.0005283 0.0005283 0.0005283 0.0005283 0.0010841 0.0010841 0.0010841

fliI rpmE ahpF ilvI cheR-2 gspC -

SO3363 SO2596 SO4680 SOA0049

20.23 20.23 20.23 20.23

0.0010841 0.0010841 0.0010841 0.0010841

-

SO4643

20.23

0.0010841

-

flagellum-specific ATP synthase FliI hypothetical protein oxidoreductase, FMN-binding ribosomal protein L31 conserved hypothetical protein alkyl hydroperoxide reductase, F subunit hypothetical protein acetolactate synthase III, large subunit hypothetical protein chemotaxis protein methyltransferase CheR conserved hypothetical protein conserved hypothetical protein general secretion pathway protein C short chain dehydrogenase family protein CBS domain protein protein-methionine-S-oxide reductase, PilB family transcriptional regulator, LysR family conserved hypothetical protein conserved hypothetical protein toxin secretion ABC transporter, ATP-binding subunit/permease protein, putative hypothetical protein

SO0430 SO4514 SO1354 SO0269 SO4509 SO4506 SO3244 SO4510 SO0105 SO4513 SO1190

-20.45 -20.45 -20.65 -22.89 -23.04 -23.65 -23.74 -23.96 -24.15 -24.51 -24.62

0.0003195 0.0003195 6.93 × 10-5 7.17 × 10-6 1.75 × 10-15 6.93 × 10-5 3.24 × 10-5 3.38 × 10-6 3.59 × 10-7 9.53 × 10-10 1.52 × 10-26

fdhB-2 flgG fdhB-1 selA -

Down-Regulated Proteins conserved hypothetical protein formate dehydrogenase, iron-sulfur subunit hemolysin protein, putative thioredoxin, putative hypothetical protein conserved hypothetical protein flagellar basal-body rod protein FlgG formate dehydrogenase, iron-sulfur subunit L-seryl-tRNA selenium transferase formate dehydrogenase, alpha subunit conserved hypothetical protein

methylation of CheY and demethlyation of CheW at longer times (Table S6 in Supporting Information). We have reported a similar motility response of S. oneidensis to hexavalent chromium,25 but did not specifically characterize PTMs in that

Transport and binding proteins Transport and binding proteins Hypothetical proteins Protein fate Biosynthesis of cofactors, prosthetic groups, and carriers Biosynthesis of cofactors, prosthetic groups, and carriers Hypothetical proteins Hypothetical proteins Protein fate Fatty acid and phospholipid metabolism Energy metabolism Cell envelope Central intermediary metabolism Transcription Hypothetical proteins DNA metabolism Hypothetical proteins Unknown function Fatty acid and phospholipid metabolism Energy metabolism Hypothetical proteins Unknown function Protein synthesis Hypothetical proteins Cellular processes Hypothetical proteins Amino acid biosynthesis Hypothetical proteins Cellular processes Hypothetical proteins Hypothetical proteins Protein fate Unknown function Unknown function Protein fate Regulatory functions Hypothetical proteins Hypothetical proteins Transport and binding proteins Hypothetical proteins Hypothetical proteins Energy metabolism Cellular processes Energy metabolism Hypothetical proteins Hypothetical proteins Cellular processes Energy metabolism Protein synthesis Energy metabolism Hypothetical proteins

data set and thus were not able to link specific movement types with the methylation dynamics of chemotactic receptors. An additional factor affecting bacterial motility could be low levels of available energy (proton motive force and/or ATP). It has Journal of Proteome Research • Vol. 8, No. 1, 2009 67

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Table 6. InsPecT p-Value Thresholds and Corresponding FDRs for Wild-Type MR-1 PTMs Total Unmodified Peptide Identifications Time point (h)

p-value

Reverse IDs

Total IDs

% FDR

0 1 3 4

0.45 0.70 0.83 0.75

733 670 759 712

68640 74244 71248 65193

2.1 1.8 2.1 2.2

a

PTM Peptide Identifications a

Reverse IDs

Total IDs

% FDR

694 621 745 706

11565 8166 8736 7415

12.0 15.2 17.1 19.0

FDR: false discovery rate.

Figure 3. Post-translationally modified peptides identified by InsPecT for strain MR-1 cultures. Each slice represents a functional category and comprises the percent of peptides for the category identified out of the total PTM peptides identified for each time point (i.e., 0, 1, 3, and 4 h).

been shown that ATP is required for functioning of chemotaxis genes (see ref 58 and references within). Under chromate stress, most of the energy is likely to be diverted to combat the effects of oxidative stress or in effluxing chromate [no known efflux system for chromate in MR-1] out of the cell thereby limiting amount of energy available for other functions such as chemotaxis. This underscores the need to carry out systematic studies of PTMs of proteins expressed under altered conditions in a microbe, in particular focusing on measurements of biological replicates for determining the reproducibility and site specificity of the modifications. Post-Translational Modification of the SO2426 Response Regulator. Response regulators are important components of two-component signal transduction pathways and are directly responsible for alterations in gene expression in response to specific external or internal stimuli. It is well-established that structural alterations via chemical modifications are necessary for response regulator activation.59-62 To develop a greater understanding of low abundance proteins such as SO2426, we focused our PTM searches of the wild-type S. oneidensis proteomic data set on identifying chemical modifications of SO2426 residues. Without considering PTMs, we could only identify 8-14 signal transduction proteins such as SO2426 at any given time point. However, by including differential 68


chemical modifications in the InsPecT search, the identification rate of signal transduction proteins increased to at least 11-18 proteins being identified over the time course. The SO2426 protein was not detected in the absence of chromate challenge (0 h time point data) using our label-free LC-MS/MS approach, even when a search for possible PTMs was applied to the data set. Following addition of Cr(VI) to wild-type S. oneidensis cultures, SO2426 was identified (with and without PTMs) in the temporal proteome data sets acquired. SO2426 was identified with 2 to 3 peptides in S. oneidensis cultures sampled at the 1 h postchromate time increment, and for the 3 h time point data, a total of six peptides passed the p-value filter thresholds used for InsPecT. At 4 h, when the chromate present in the medium was completely reduced by the wild-type, the number of peptides detected for SO2426 decreased to three. PTMs detected at the 3 and 4 h time points during chromate challenge consisted of oxidation occurring primarily on the Cys-8 and Met-11 residues of the protein. Figure 4 presents the tandem mass spectra of a heavily modified peptide for SO2426 from the 3 h time point, and highlights not only the specific methionine target, but also reveals some adjacent modification sites. There are also a variety of methylations observed, but there does not appear to be a noticeable correlation with chromate exposure. At this level of investiga-


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Figure 4. A peptide MS/MS from SO2426 with confirmed modifications detected with p-value of 0.8 and a match quality (MQ) score of 1.62. The N-terminal peptide from SO2426 (MILILVWC+48LEM+32SR+84) has two oxidation events and a diacetylation event. Fragment ions y4, y7, y8, and y10 confirm the diacetylated arginine residue, while fragment ions b8, b9, and b*10 confirm the triply oxidized methionine. All three modifications are present in fragment ions y7, y8, and y10.

tion, it is unclear what role the modifications might play in the functional activity of the SO2426 protein in vivo. It is possible that the oxidations might simply represent a level of protein damage as a result of chromate exposure rather than any contribution to a specific functional role.

Conclusions The comparative LC-MS/MS study described in this paper was employed to measure protein abundance dynamics on a genome-wide scale between a response regulator (SO2426)deficient strain of S. oneidensis MR-1 and the parental strain prior to and during chromate challenge and subsequent transformation. Our overall goal was to characterize temporal alterations in global protein machinery as a direct or indirect consequence of targeted deletion of the so2426 gene, which was demonstrated in our previous work to be involved in the cellular response to acute chromate stress. Although the ∆so2426 mutant was impaired in its capacity to reduce extracellular Cr(VI) compared to the wild-type, the total number of nonredundant proteins identified in the two strains was relatively comparable across the four time points of analysis (i.e., 0, 1, 3, and 4 h postchromate addition). However, there were noteworthy differences in the subsets of differentially expressed proteins between the mutant and wild-type, which suggested a potential functional role of SO2426 linked to the regulation (direct or indirect) of iron uptake systems. In the absence of chromate, a siderophore biosynthesis protein (SO3032) and a TonB-dependent receptor (SO4743) in particular were measured at extremely low abundance levels in the ∆so2426 mutant in contrast to the wild-type. The temporal expression profiles of the siderophore biosynthesis protein AlcA (encoded in an operon along with SO3031 and SO3032) and a ferric alcaligin siderophore receptor (SO3033) in the mutant remained down-regulated across the chromate challenge time course, suggesting that SO2426 may be involved in the activation of genes codifying the siderophore-mediated iron transport system in S. oneidensis. The differential expression observed

for certain iron-requiring energy metabolism proteins could be explained by an indirect effect associated with perturbed intracellular iron availability in the mutant. Involvement of iron in nonenzymatic chromate reduction has been reported earlier (see ref 63 and references within). Although an excess of free intracellular Fe(II) is unlikely due to its participation in hydroxyl radical-generating Fenton reactions, some of the available free Fe(II) for protein incorporation and as a cofactor for DNAbinding activity of Fur (the dominant sensor of Fe availability in bacteria39) may be reacting with chromate, thereby depleting intracellular iron pools and disrupting Fe homeostasis. It is not clear based on the temporal proteomics data how perturbed iron availability is linked to the impaired chromate removal rate exhibited by the ∆so2426 mutant, other than functional iron acquisition systems seem to play an important role in the ability of S. oneidensis MR-1 to effectively cope with chromate stress. By mining the dynamic proteome data set for posttranslational modifications present on the chemotaxis CheY and CheW proteins, as well as the SO2426 protein, we identified a number of PTM targets that warrant further study. Such modifications appear to influence the activity of response regulators in signal transduction pathways and study of such modifications will significantly enhance our knowledge of response regulation pathways in microbes.

Acknowledgment. M. R. Thompson acknowledges support from the University of Tennessee (Knoxville)-ORNL Graduate School of Genome Science and Technology. We thank Dr. Yuan (Susie) Dai and Carlee McClintock for critical reading of the manuscript and excellent suggestions. This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-06ER64163. Oak Ridge National Laboratory is managed by University of Tennessee-Battelle LLC for the Department of Energy under contract DOE-AC05-00OR22725. Supporting Information Available: Protein expression ratio for mutant vs WT at 0 h, and 1, 3, and 4 h following Journal of Proteome Research • Vol. 8, No. 1, 2009 69

research articles chromate shock, combined mutant/WT protein expression ratios at different time points during chromate shock experiment, and PTM results using Inspect with the WT MR-1 strain of S. oneidensis prior to chromate exposure. This material is available free of charge via the Internet at http://pubs.acs.org.

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Journal of Proteome Research • Vol. 8, No. 1, 2009 71

Comparative Temporal Proteomics of a Response Regulator

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