Lin et al. 10.1073/pnas.0603974103. |
Fig. 5. Multiple alignment of ArsD homologues. Representative ArsD homologues are from: E. coli plasmid R773 (accession no. U13073); Salmonella typhimurium plasmid R64 (U38947); Klebsiella oxytoca plasmid pMH12 (AF168737); Acidiphilium multivorum plasmid pKW301 (AB004659); E. coli plasmid R46 (NP_511237); Shewanella sp. ANA-3 (AYA271310.1); Rhodospirillum rubrum (ZP_00269301); Rhodopirellula baltica SH 1 (CAD76333); Listeria innocua Clip11262 (NP_569188); Bacteroides thetaiotaomicron VPI-5482 (NP_809714); Bacillus sp. CDB3 (AAD51848); and Halobacterium sp. NRC-1 megaplasmid pNRC100 (NC_001869). Cysteine residues are indicated. The multiple alignment was calculated with CLUSTAL W [Thompson JD, Higgins DG, Gibson TJ (1994) Nucleic Acids Res 22:4673-4680].
Fig. 6. ArsD enhances the activity of the ArsAB pump in vivo. (A) Coexpression of the arsD gene with arsAB increases arsenite resistance. Cells of E. coli strain AW3110 (DarsRBC) harboring vector plasmids pSE380 and pACBAD (○), pSE380 and pACBAD-D (arsD) (Ñ), pSE-B (arsB) and pACBAD (◊), pSE-B (arsB) and pACBAD-D (arsD) (□), pSE-AB (arsAB) and pACBAD (●), and pSE-AB (arsAB) and pACBAD-D (arsD) (▼) were grown in LB medium overnight. The cells were then diluted 50-fold into LB medium containing 10 mM sodium arsenite. The absorbance at 600 nm was measured after incubation at 37°C for the indicated times. The values in each plot are the mean of three growth assays. The error bars represent the standard deviation of the mean calculated by using SigmaPlot 9.0.
Fig. 7. ArsD does not affect the level of ArsA. Samples were analyzed by SDS/PAGE, stained with Coomassie blue (A), and immunoblotted with anti-ArsA (B) or anti-ArsD (C). Lane 1, standard proteins; lane 2, 2 mg each of purified ArsA and ArsD; lane 3, cells from 0.2 ml of a culture (A600 nm = 1) of E. coli strain AW3110 (DarsRBC) harboring vector plasmids pSE380 and pAC-BAD; lane 4, cells from 0.2 ml of a culture (A600 nm = 1) of AW3110 cells expressing plasmids pSE-AB (arsAB) and pAC-BAD; lane 5, cells from 0.2 ml of a culture (A600 nm = 1) of expressing plasmids pSE-AB (arsAB) and pAC-BAD-D (arsD). The positions of ArsA and ArsD are indicated by arrows.
Table 1. ars operons with arsA and arsD genes
ars operons | Swiss-Prot/TrEMBL accession nos. | |
| arsD | arsA |
Plasmid-encoded (Organism, plasmid) |
|
|
Escherichia coli , R773 | P46003 | P08690 |
Salmonella typhimurium , R64 | Q8L253 | Q8L248 |
Klebsiella oxytoca , pMH12 | Q9KJI4 | Q9KJI3 |
Acidiphilium multivorum , pKW301 | O50592 | O50593 |
Escherichia coli , R46 | P52148 | P52145 |
Rhodococcus erythropolis, pREL1 | Q3L9K6 | Q3L9K5 |
Rhodococcus erythropolis, pBD2 | Q6XN06 | Q6XN05 |
Listeria innocua, pLI100 | Q926M4 | Q926M2 |
Staphylococcus saprophyticus, pSSP1 | Q49UG6 | Q49UG7 |
Bacillus cereus, pBc10987 | Q74NT7 | Q74NT8 |
Halobacterium salinarium , pNRC100 | O52028 | O52027 |
Genomic |
|
|
Shewanella sp. W3-18-1 | Q2X358 | Q2X359 |
Shewanella sp. ANA3 | Q366A2 | Q366A1 |
Shewanella putrefaciens CN-32 | Q2ZUN1 | Q2ZN2 |
Leptospirillum ferriphilum, TnLfArs | Q2LMN6 | Q2LMN5 |
Alcaligenes faecalis NCIB 8687 | Q6WB30 | Q6WB29 |
Methylobacillus flagellatus KT | Q1H177 | Q1H178 |
Azoarcus sp. EbN1 | Q5P148 | Q5P147 |
Dechloromonas aromatica RCB | Q47CR3 | Q47CR4 |
Acidithiobacillus caldus TnAtcArs | Q3T561 | Q3T560 |
| Q3T559 | Q3T558 |
Rhodoferax ferrireducens T118 | Q21S88 | Q21S89 |
Photobacterium profundum SS9 | Q6LRX4 | Q6LRX3 |
Photobacterium profundum 3TCK | Q1Z8S1 | Q1Z8S2 |
Mycobacterium vanbaalenii PYR-1 | Q25WE1 | Q25WE1 |
Psychromonas sp. CNPT3 | Q1ZFE2 | Q1ZFE1 |
Mycobacterium flavescens PYR-GCK | Q277W2 | Q277W3 |
Burkholderia vietnamiensis G4 | Q4BLB4 | Q4BLB3 |
Alkalilimnicola ehrlichei MLHE-1 | Q34YA7 | Q34YA8 |
Rhodopirellula baltica SH 1 | Q7ULE4 | Q7ULE5 |
delta proteobacterium MLMS-1 | Q1NIX3 | Q1NPV7 |
Rhodospirillum rubrum ATCC 11170 | Q2RUE6 | Q2RUE7 |
Clostridium sp. OhILAs | Q1EZS8 | Q1EZS7 |
Clostridium beijerincki NCIMB 8052 | Q2WLD2 | Q2WLD1 |
Magnetospirillum magneticum AMB-1 | Q2W689 | Q2W688 |
Desulfitobacterium hafniense Y51 | Q24PV2 | Q24PV1 |
Desulfitobacterium hafniense DCB-2 | Q423V8 | Q423V9 |
Syntrophomonas wolfei subsp. Goettingen | Q3G9J8 | Q3G9J9 |
Bacteroides thetaiotaomicron VPI-5482 | Q8ABJ3 | Q8ABJ4 |
Clostridium phytofermentans ISDg | Q1FPR6 | Q1FPR4 |
Lactobacillus plantarum WCFS1 | Q6LWG6 | Q6LWG5 |
Staphylococcus epidermidis RP62A | Q5HKC1 | Q5HKC0 |
Staphylococcus epidermidis ATCC 12228 | Q8CQF1 | Q8CQF2 |
Bacillus sp. CDB3 | Q9RA90 | Q9RA89 |
Bacillus sp. MB24 TnARS1 | Q5Q1Q6 | Q5Q1Q5 |
Geobacter uraniumreducens Rf4 | Q2DL37 | Q2DL38 |
Staphylococcus haemolyticus JCSC1435 | Q4LAA4 | Q4LAA5 |
Haloquadratum walsbyi | Q18H25 | Q18H24 |
Alkaliphilus metalliredigenes QYMF | Q3CDN9 | Q3CDP0 |
Q3CDP1 | ||
Q3C8Z0 | Q3C8Z1 | |
Q3C8Z2 | ||
Supporting Materials and Methods
Plasmid Construction and Yeast Two-Hybrid Analysis.
Plasmids with the arsDAB, arsAB, arsB, and arsD genes were constructed as follows. Plasmid pET28a was changed to pET28a1 by replacing the HindIII site with a StuI site and introduction of a HindIII site behind the XbaI site in the multiple cloning region by PCR. The arsAB genes were excised from plasmid pAlterAB1 as a HindIII-EcoRI fragment and ligated with HindIII-EcoRI-digested pET28a1, generating plasmid pET-AB. Similarly, the arsB gene was excised from pAlterAB2 into the HindIII and EcoRI sites of pET28a1, generating plasmid pET-B. An XbaI-HindIII-truncated arsD gene was made by PCR, digested with both restriction enzymes, and ligated with similarly digested pET-AB, generating plasmid pET-DAB. For construction of a plasmid with a full-length arsD gene, the truncated arsDD119-120 gene on pArsD6HD119-120 (1) was modified by introduction of an additional XbaI site immediately following the EcoRI site, the sequence for Cys-119 and Cys-120 was inserted by PCR, and the gene was isolated as a EcoRI-BamHI fragment and ligated with EcoRI-BamHI-digested pSE380, producing plasmid pSE-D. This plasmid was transformed into E. coli BL21(DE3) for purification of nontagged full-length ArsD. The arsDAB, arsAB and arsB genes were cloned into plasmid pSE-D by using the XbaI and XhoI sites from pET-DAB, pET-AB, and pET-B, generating plasmids pSE-DAB, pSE-AB, and pSE-B, respectively. The pSE-DAB and pSE-AB plasmids were used for molecular competition assays. The arsD gene also was cloned as follows. A 380-bp PCR product containing the PBAD promoter region from pBAD/Myc-HisA was cloned into pACYC184 by using the BclI and EcoRI sites, generating pACBAD. The full-length arsD gene was PCR-cloned into this plasmid by using the NcoI and EcoRI sites, generating pACBAD-D, which was cotransformed with pSE-AB or pSE-B into E. coli strain AW3110 and used for transport assays.For use in yeast two-hybrid assays, plasmids were constructed as follows. The arsR gene was engineered with an EcoRI site at the 5' end, followed immediately by a NcoI site and a BamHI site after the stop codon at the 3' end by PCR and cloned into the GAL4 DNA-binding domain (BD) fusion plasmid pGBT9 through EcoRI and BamHI sites and the activation domain (AD) fusion plasmid pACT2 through NcoI and BamHI sites, generating pGBT-R and pACT-R, respectively. The arsA, arsC, and arsD genes were cloned similarly by using the NcoI and BamHI sites on pGBT-R and pACT2, generating pGBT-X (BD-X) series and pACT-X (AD-X) series plasmids. For construction of the N-terminal maltose-binding protein (MBP)-ArsD chimera, an EcoRI-SalI fragment containing the entire arsD gene was cloned from pGBT-D into pMAL-c2X plasmid, generating plasmid pMAL-D.
A GAL4-based yeast two-hybrid system (2) (CLONTECH) was used to determine protein-protein interactions. AH109, a GAL4-activating HIS3 reporter yeast strain, was cotransformed with ars gene-fused BD-X series and AD-X series plasmids. The transformed cells were cultured overnight in SD medium at 30°C and washed, suspended, and adjusted to A600 of 1 in 20 mM Tris·HCl, pH 7.5. Portions (1 ml) were inoculated on SD agar plates lacking histidine without or with 0.1 mM sodium arsenite in serial 10-fold dilutions and incubated at 30°C for 2-3 days. As a positive control, pVA3 (BD-p53) was expressed with pTD1 (AD-T antigen); as a negative control, vector plasmid pGBT9 was expressed with pACT2.
Protein Purification.
Cells bearing the indicated plasmids were grown in LB medium overnight at 37°C and diluted 50-fold into 1 liter of LB containing 0.1 mg/ml ampicillin or 40 mg/ml kanamycin. Proteins were expressed by induction with 0.3 mM IPTG at A600 of 0.6-0.8 for 3 h. Wild-type ArsD was purified from E. coli BL21(DE3) bearing plasmid pSE-D. Induced cells were harvested by centrifugation and washed once with a buffer containing 20 mM Tris·HCl (pH 7.5), 0.2 M NaCl, 1 mM EDTA, and 5 mM DTT (buffer A). The cells were suspended in 5 ml of buffer A per g of wet cells and lysed by a single passage through a French press at 20,000 psi. Diisopropyl fluorophosphate was added at 2.5 ml/g of wet cells immediately after lysis. Unbroken cells and membranes were removed by centrifugation at 10,000-150,000 ´g for 1 h at 4° C. ArsD was purified as described (1) and stored at -80°C until used.The MBP-ArsD chimera was purified from BL21(DE3)/pMAL-D. Cytosol was applied to a 1 ´ 10 cm amylose column (New England Biolabs) preequilibrated with buffer A. The column was washed with 120 ml of buffer A, and the chimeric protein was eluted with buffer A containing 10 mM maltose. MBP-ArsD-containing fractions were identified by SDS/PAGE, pooled, concentrated, and stored in small aliquots at -80°C until use. ArsA with a six-histidine tag at the C terminus was expressed in BL21(DE3) harboring plasmid pSE-AB. The protein was purified as described (3) and stored at -80°C until use. Protein concentrations were determined from the absorption at 280 nm by using calculated molar extinction coefficients (4).
Fluorescence Measurements and ATPase Assays.
Fluorescence spectra were determined in a Jasco FP750 fluorometer at 20°C. Tryptophan fluorescence was excited at 285 nm, and emission was scanned between 300 and 400 nm. Dissociation of Sb(III) from ArsD with a six-histidine tag was initiated by diluting the protein >100-fold and monitored from the resulting increase in the protein fluorescence of ArsD (5). Stopped-flow fluorescence spectroscopy was used to time-resolve the interaction of the ArsD-Sb(III) complex with ArsA. In these assays, the Trp-159 single tryptophan derivative of ArsA was used to reduce background fluorescence (6). Time-resolved fluorescence measurements were carried out in an Applied Photophysics (London, U.K.) SX.18MV stopped-flow instrument, operated at 20°C. For measurements of the change in tryptophan fluorescence, the samples were excited at 285 nm and selected with a monochromator, and the emission was monitored at wavelengths above 335 nm by using a cut-off filter. Routinely, equal volumes of the reactants were mixed together in the stopped-flow instrument, and concentrations are for the mixing chamber.ATPase activity was estimated by using a coupled assay (7). MBP-ArsD was buffer-exchanged into 50 mM Mops-KOH, pH 7.5/0.25 mM EDTA and added at 3 mM, final concentration, into the same buffer plus 5 mM ATP, 1.25 mM phosphoenolpyruvate, 0.25 mM NADH, and 10 units of pyruvate kinase and lactate dehydrogenase with or without potassium antimonyl tartrate or sodium arsenite. ArsA was added at 0.3 mM, final concentration. The reaction was initiated by addition of 2.5 mM MgCl2 and measured at 340 nm at 37°C. The linear steady-state rate of ATP hydrolysis was used to calculate specific activities.
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7. Vogel G, Steinhart R (1976) Biochemistry 15:208-216.