Cellular Response of Sinorhizobium sp. Strain A2 during Arsenite Oxidation

Arsenic (As) is a widely distributed toxic element in the environment and microorganisms have developed resistance mechanisms in order to tolerate it. The cellular response of the chemoorganotrophic arsenite (As[III])-oxidizing α-Proteobacteria, Sinorhizobium sp. strain A2, to arsenic was examined in the present study. Several proteins associated with arsenite oxidase and As resistance were shown to be accumulated in the presence of As(III). A shift in central carbon metabolism from the tricarboxylic acid pathway to glyoxylate pathway was also observed in response to oxidative stress. Our results revealed the strategy of the As(III)-oxidizing Sinorhizobium strain to mitigate arsenic toxicity and oxidative damage by multiple metabolic adaptations.

G-250 staining and gels were digitized using Chemidoc XRS system (Bio-Rad). Image analysis was performed with the Progenesis SameSpots software (Nonlinear Dynamics, Durham, NC) on the two image sets in duplicate from each of five independent cultures. Spot quantity values were normalized in each gel by dividing the raw quantity of each spot by the total quantity of all the spots included in the gel, and normalized spot values were statistically analyzed by one-way ANOVA.

Sample preparation for nanospray LC-MS/MS analyses
Protein spots exclusively expressed in the presence of As(III) were identified, excised from 2D-gel and destained in 30% (v/v) acetonitrile containing 25 mM NH 4 HCO 3 . In-gel digestion was performed as previously described (5). Briefly, the gel pieces were reduced in 25 mM NH 4 HCO 3 containing 10 mM DTT at 56°C for 60 min, then alkylated in 55 mM iodoacetamide in the dark at room temperature for 45 min, followed by the digestion with 10 ng μl -1 trypsin (Promega, Madison, WI) at 37°C overnight. The digested peptides were extracted with the solution consisted of 50% (v/v) acetonitrile and 5% (v/v) arifluoroacetic acid (TFA).
For 1D LC-MS/MS analysis (Table S1), protein extracts were prepared as described above for 2DE gels and precipitated with four volumes of ice-cold acetone at -25°C overnight. Precipitated proteins were separated by centrifugation (10 min, 12000  g, 4°C), washed twice with ice-cold acetone and homogenized in 50 mM NH 4 HCO 3 . Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad). The proteins were reduced with DTT at a ratio of 1:20 (w/w) at 56°C for 30 min, alkylated with iodoacetamide at a ratio of 1:4 (w/w) in the dark at room temperature for 30 min.
The protein mixture digested with trypsin at a ratio of 1:20 (w/w) at 37°C for 16 h. The digestion was stopped by the addition of 1 μl of formic acid (2). Peptide mixtures were desalted using Sep-Pack C18 cartridge (Waters, Milford, MA) and concentrated to ~100 µl by vacuum centrifugation.

Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses
LC-MS/MS analysis was performed on an LCQ Advantage mass spectrometry system (ThermoFinnigan) coupled to Paradigm MS4 HPLC (Michrom Bioresources) and a Magic C18 column Search parameters were employed from previously reported SEQUEST filtering criteria for validating the identifications (2, 3): ∆Cn > 0.1, Xcorr of 1.9, 2.2 and 3.75 for +1, +2 and +3 charge states, respectively. Furthermore, only those proteins with more than two unique peptides or a single unique peptide that has at least seven amino acids and had a minimum of three successive b or y series ions were considered as reliably identified (2,4).
For 1D LC-MS/MS analysis, semi-quantitative protein abundance was determined as described previously (1, 2) using LC-MS/MS results from triplicate cultures for each treatment. Briefly, the protein abundance index (PAI) was calculated as: where CP is the total peptides count for each detected protein and OP is the number of observable peptides. The OP was calculated by in silico trypsinization of the protein by using the IPEP only proteolysis (http://ipep.moffitt.org/serchProtein.cgi). Protein abundance estimate (emPAI) was then calculated as follows (Table S1). emPAI=10 PAI -1 Supplemental figure S1. Detection of arsC and acr3 transcripts in Sinorhizobium sp. strain A2 growing in the presence of 10 mM As(III). Arrows indicate the expected amplification products. Total RNA was prepared from the cells grown with 10 mM As(III) and harvested at 24, 48, and 120 hrs as described in the manuscript for qRT-PCR. RT-PCR was performed using the Access RT-PCR system (Promega, Madison, WI) and the reaction mixture (50 µl) contained 1 µM of each primer and ~50 ng of extracted RNA. Control reactions were performed without addition of reverse transcriptase to verify the absence of DNA in the RNA preparations. Primers for arsC (Gene ID: 5319357) and acr3 (Gene ID: 5319356) were designed based on those sequences from Sinorhizobium medicae WSM419 using the Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi). The primer sets arsC52F (5'-TCGCGGAACACCTTGGCTATG) and arsC384R (5'-TATGAAAGCGCCCTTCTGCTCTTG), and acr755F (5'-TGCCCATCCTCATCCAGGTCTAC) and acr986R (5'-ATCAAGACCACGGACAGCATCAC) were used to amplify arsC and acr3, respectively.