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1 State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2 Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
Si-Qi Liu
siqiliu{at}genomics.org.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Three supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and synthesis of phospholipids (Ordóñez et al., 2005). In many organisms, cells detoxify arsenate by an arsenate-resistance system (ARS). This ARS includes a reductase that reduces arsenate to arsenite, which is subsequently extruded from cells. The prokaryotic arsenate reductases can be classified into two families (and the eukaryotic arsenate reductase represents the third family): family I arsenate reductases need glutaredoxin as the source of reducing equivalents, and are represented by the arsC products from Escherichia coli plasmid R773 (Chen et al., 1986) and some other Gram-negative bacteria. Family II arsenate reductases need thioredoxin as the source of reducing equivalents and are structurally related to low-molecular-mass protein tyrosine phosphatases. Examples of the family II arsenate reductases are the arsC products of Staphylococcus aureus and Bacillus subtilis (Bennett et al., 2001; Ji & Silver, 1992) (a phylogentic tree indicating functionally identified and putative arsenate reductases is shown in supplementary Fig. S1, available with the online version of this paper).
arsC and other arsenate-resistance genes are usually found in one operon (the ars operon) located on the chromosome or a plasmid. In the case of E. coli plasmids R773 and R46, the ars operon consists of five genes, arsR, arsD, arsA, arsB and arsC, that respectively encode arsenate-resistance regulator, arsenate chaperone, efflux pump subunit, arsenate permease and arsenate reductase (Chen et al., 1985; Bruhn et al., 1996). However, the ars operons of plasmids pI258 and pSX267 from Staphylococcus species (Ji & Silver, 1992; Rosenstein et al., 1992) and of the E. coli chromosome (Diorio et al., 1995) consist of only three genes, arsR, arsB, and arsC. Common to all ars operons are the genes arsR, arsB, and arsC, which encode regulator, arsenite efflux pump and arsenate reductase, respectively (Rosen et al., 1988; San Francisco et al., 1990; Tisa & Rosen, 1990; Wu & Rosen, 1991; Gladysheva et al., 1994; Chen et al., 1986; Chen & Rosen 1997).
After exposure to arsenate, the ars genes are transcribed and the corresponding proteins synthesized, leading to arsenate resistance (Wang et al., 2006; Muller et al., 2007). More recently, enhanced expression of heat-shock proteins in the presence of arsenate was found in the acidophilic archaeon Ferroplasma acidarmanus by application of two-dimensional PAGE (2-DE) (Baker-Austin et al., 2007). Further, global analysis of cellular responses to arsenite revealed that arsenite exposure results in an oxidative-stress-like response in Pseudomonas aeruginosa (Parvatiyar et al., 2005).
Comamonas sp. strain CNB-1 was isolated from activated sludge and uses 4-chloronitrobenzene as sole carbon and energy source (Wu et al., 2005). Recently, plasmid pCNB1, encoding the 4-chloronitrobenzene degradation pathway was sequenced, and analysis of the nucleotide sequence revealed several genes that are putatively involved in arsenate resistance (Ma et al., 2007). In this study, the arsenate resistance and global responses of strain CNB-1 to arsenate exposure were studied at genetic and proteomic levels.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Comamonas sp. strain CNB-1 was cultivated in Luria–Bertani (LB) medium with or without 100 mM sodium arsenate. Cells were harvested after 24 h cultivation. All E. coli strains were cultivated aerobically at 37 °C in LB medium. When required, ampicillin was added to the medium at a final concentration of 100 µg ml–1.
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). For pCNB1 isolation, a modified alkaline lysis method was used (Sinnett et al., 1998). PCR amplification of putative ars genes was performed with the primers described in Table 1
. The PCR reaction was carried out with a Biometra thermocycler by using Pfu Taq DNA polymerase (Trans, Beijing, China) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation (40 s at 94 °C), an annealing step of 40 s, and a 1–2 min elongation step at 72 °C. Annealing temperatures and elongation times were altered according to the GC content of primers and the size of targeted DNA fragments. PCR products were separated by agarose gel electrophoresis and further purified by using the agarose gel DNA fragment recovery kit (Tiangen, Beijing, China).
Determination of arsenate resistance by Comamonas sp. strain CNB-1 and complementation of E. coli mutants.
To test for growth of Comamonas sp. strain CNB-1 in the presence of arsenate, the strain was pre-cultured in LB broth for 24 h and this pre-culture was used to inoculate fresh LB medium containing different concentrations of sodium arsenate (0–200 mM). Growth was monitored by measuring the OD600 with a spectrophotometer.
Complementation of arsenate resistance in E. coli mutants WC3110 and AW3110 was performed with newly constructed plasmids carrying the different ars genes from Comamonas sp. strain CNB-1, which were cloned in the vector pSE380 (Table 1). Growth of recombinant E. coli strains was determined in LB medium as described above for Comamonas sp. strain CNB-1. Expression of targeted ars genes was induced with 0.5 mM IPTG and various concentrations of sodium arsenate. The E. coli cultures were incubated at 37 °C and 160 r.p.m. for 24 h.
RNA isolation and RT-PCR.
For RNA isolation, Comamonas sp. strain CNB-1 was cultivated in LB broth containing 100 mM sodium arsenate. Total RNA was isolated using Trizol reagent kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The extracted RNA was dissolved in 50 µl water. To eliminate any genomic DNA, this RNA preparation was incubated with 10 U DNase and 40 U RNase inhibitor (Takara, cat. no. D2310A) for 30 min at 37 °C. To obtain cDNAs, the above preparations (8 µl, containing 2 µg RNA) were mixed with 4 µl of 10 µM primer PBRTR for arsPCom-arsBCom or BCRTR for arsBCom-arsCCom (Table 1) in final volumes of 12 µl. To facilitate reverse transcription, any RNA secondary structures were eliminated by incubation at 70 °C for 5 min, and pairing between RNA templates and primers was stimulated by quickly placing the mixtures on ice. cDNAs were synthesized in reaction mixtures (total volume 21 µl) containing the above-prepared mixture (12 µl), 4 µl reaction buffer (Promega), 1 µl of 10 mM dNTP, 1 µl RNase inhibitor, 2 µl of 0.5 mM MgCl2 and 1 µl ImProm-II reverse transcriptase (Promega). The mixtures were incubated for 60 min at 42 °C and reaction was terminated by incubation at 70 °C for 15 min. The cDNAs synthesized were used for amplification of arsPB or arsBC fragments with PCR. PCRs were carried out using Taq DNA polymerase (Promega) and primers PBRTF/PBRTR for arsPCom-arsBCom and BCRTF/BCRTR for arsBCom-arsCCom, respectively (Table 1).
2-DE and image analysis.
Comamonas sp. strain CNB-1 was first grown aerobically overnight in LB broth and then was inoculated into 100 ml fresh LB broth with and without 100 mM sodium arsenate for 24 h. After harvest, the cells were washed twice with 50 mM Tris/HCl (pH 7.4) and resuspended in 1.2 ml lysis buffer (8 M urea, 4 % CHAPS, 2 % Bio-Lyte) followed by sonication on ice. The supernatant generated from centrifuging at 7000 g was collected and stored at –20 °C for proteomic analysis. Protein concentration was determined by the Bradford method with BAS calibration daily (Bradford, 1976).
The protein extracts were treated with DNase I and RNaseA at final concentrations of 1 mg ml–1 and 0.25 mg ml–1, respectively, and at 4 °C for 30 min. Approximately 300 µg proteins was loaded onto the 2-DE system. Briefly, first-dimension IEF was performed on 13 cm pH 4–7 strips or 11 cm pH 6–11 strips. The strip gel (pH 4–7; GE Healthcare) was rehydrated overnight in rehydration solution (8 M urea, 4 % CHAPS, 50 mM DTT, 0.5 % IPG buffer pH 4–7) containing the samples. IEF was performed in Ettan IPGphor II (GE Healthcare) with a stepwise programme at 500 V for 1 h, 1000 V for 1 h, and 8000 V for 55 000 V h. The strip gel (pH 6–11) was rehydrated overnight in modified buffer (8 M urea, 4 % CHAPS, 10 % 2-propanol, 5 % glycerol, 1.5 % hydroxyethyldisulfide (HED) and 1 % IPG buffer pH 6–11). IEF was carried out by the stepwise programme at 150 V for 0.5 h, 300 V for 3 h, 600 V for 2 h, and 3500 V for 35 000 V h. The IEF strips were consecutively equilibrated with reducing buffer containing 6 M urea, 375 mM Tris/HCl pH 8.8, 30 % (v/v) glycerol, 2 % (w/v) SDS and 2 % (w/v) DTT for 15 min, and with alkylating buffer containing 6 M urea, 375 mM Tris/HCl pH 8.8, 30 % (v/v) glycerol, 2 % (w/v) SDS and 2.5 % (w/v) iodoacetamide for 15 min. The second-dimension electrophoresis was performed on a 13 % polyacrylamide gel using an SE600 Ruby device (GE Healthcare), which was run at 15 mA per gel for 30 min, and then at 30 mA per gel until the bromophenol blue dye front reached the gel bottom. The protein spots on the 2-DE gels were visualized with Coomassie blue R-250 (Bio-Rad). To enable statistical evaluation of 2-DE images, all the samples were run in triplicate.
The stained 2-DE gels were scanned using UMAX Powerlook 2100XL at a resolution of 400 d.p.i. Image analysis was performed with ImageMaster 2D Platinum 6.0 software (GE Healthcare). The relative volume (vol%) for each spot was normalized with the total spot volume at each gel. Student's t-test was adopted to evaluate the significantly differential spots between the control and experimental gels (P<0.05). A twofold change in the relative spot volumes was defined as the threshold for significant difference in these 2-DE spots.
Tryptic in-gel digestion.
The differential 2-DE spots were carefully excised and destained overnight at 37 °C in a solution of 25 % ethanol and 7 % acetic acid. The gel particles were reduced with 10 mM DTT in 25 mM ammonium bicarbonate at 56 °C for 1 h and alkylated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate in the dark at room temperature for 45 min in situ. After complete drying, the gel pieces were digested overnight at 37 °C in 2–3 µl modified trypsin (10 ng µl–1, sequencing grade; Roche Diagnostics). The digestion reaction was stopped by addition of trifluoroacetic acid (TFA) at a final concentration of 0.1 %.
Protein identification by MALDI-TOF/TOF MS.
One microlitre of the digestive mixture was loaded on an Anchorchip target (Bruker Dalton) and allowed to dry completely. Then 0.1 µl matrix solution (4 mg ml–1 
-cyano-4-hydroxycinnamic acid, CHCA) was added to the target and mixed with the digested peptides. After washing with 0.1 % TFA, the Anchorchip was delivered to MALDI-TOF/TOF MS for protein identification. Mass spectra and tandem mass spectra were obtained on an Ultraflex TOF/TOF mass spectrometer (Bruker Dalton). Positively charged ions were analysed in the reflector mode, using delayed extraction. The mass spectrometer was operated under 19 kV accelerating voltage in the reflection mode and an m/z range of 600–4000. Typically, 100 shots were accumulated per spectrum in MS mode and 400 shots in MS/MS mode. The spectra were processed using the FlexAnalysis 2.2 and BioTools 2.2 software tools.
Based upon mass signals, protein identification was performed using the Mascot software (http://www.matrixscience.com) to search the draft genome of Comamonas sp. strain CNB-1 on a local database, which was generated by shotgun approach. The draft genome contained 544 contigs. ORFs were predicted online by using the web version of the software GeneMarkS (http://exon.gatech.edu/GeneMark/). The total predicted ORFs were formatted in FASTA and a specific protein database of strain CNB-1 was constructed. The mass signals were also taken for search against the NCBInr database with bacteria as taxonomy. The following parameters were used for database searches: monoisotopic mass accuracy <100 p.p.m., missed cleavages 1, carbamidomethylation of cysteine as fixed modification, oxidation of methionine, N-terminal pyroglutamylation (peptide) and N-terminal acetylation (protein) as variable modifications. In MS/MS mode, the fragment ion mass accuracy was set to <0.7 Da.
Sequence analysis.
Multiple sequence alignment for arsenate reductases was performed with CLUSTAL_X (Jeanmougin et al., 1998) and sequence identity was calculated with BioEdit (Tippmann, 2004).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Four genes, arsRCom, arsPCom, arsBCom and arsCCom, that were putatively involved in arsenate resistance were consecutively located on a putative transposon (Fig. 1a). Protein homology searches and sequence alignment indicated that (1) ArsRCom had moderate sequence identities to the functionally identified ArsRs of Acidithiobacillus ferrooxidans (48 %) and Acidithiobacillus caldus (38 %); (2) ArsBCom had high identities to the ArsBs of E. coli R773 (73 %), P. aeruginosa (76 %), and A. ferrooxidans (64 %); (3) ArsCCom showed high sequence identities to the arsenate reductases belonging to family I, such as ArsCs in E. coli R773 (65 %), P. aeruginosa (80 %) and Sinorhizobium meliloti (71 %) (see also supplementary Fig. S1); (4) ArsPCom, however, had significant sequence identity to the putative low-molecular-mass tyrosine phosphatases from Acidocorax sp. JS42 (73 %), P. aeruginosa (64 %), and Burkholderia multivorans (63 %) – it also showed moderate identities (35.4–56.4 %) to the family II arsenate reductases from Acidithiobacillus ferrooxidans, Acidithiobacillus caldus and Herminiimonas arsenicoxydans (see also supplementary Fig. S1); and (5) there were no ArsA, ArsD and ArsO homologues (Fig. 1b), which have been identified in other arsenate-resistant bacteria near the ars genetic cluster of strain CNB-1.
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). It was more resistant to arsenate than Mycobacterium smegmatis (25 mM), Staphylococcus aureus (50 mM), B. subtilis (75 mM) and E. coli (100 mM) (Ordóñez et al. 2005). The arsenate resistance of strain CNB-1 was clearly related to the occurrence of pCNB1 within cells. Compared to strain CNB-1, strain CNB-2, which had lost pCNB1, was less resistant but it retained some resistance(
60 % growth at 60 mM arsenate) (Fig. 2a). This result suggests that strain CNB-1 also has a chromosomic ars operon.
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). Furthermore, when the genes arsBCom and arsCCom were concomitantly cloned and expressed in the ars-operon-deficient E. coli AW3110, which is deficient in arsenate reductase and arsenite permease, the resulting strain had acquired resistance to arsenate (data not shown). These results support that the arsBCom and arsCCom of strain CNB-1 encode arsenite permease and arsenate reductase, respectively. Similarly, the gene arsPCom complemented arsenate resistance of E. coli WC3110, suggesting that ArsPCom functioned as an arsenate reductase (Fig. 2c). Further, concomitant cloning of ars(PB)Com in E. coli AW3110 restored arsenate resistance (data not shown). The above results demonstrated that strain CNB-1 has two arsenate reductases (ArsCCom and ArsPCom).
arsPCom and arsCCom are both transcribed in the presence of arsenate and belong to the same transcriptional unit
To confirm that either arsPCom or arsCCom or both were functional during growth in the presence of arsenate, transcription of the two genes was examined by RT-PCR (primers used are indicated in Fig. 1a). The results clearly showed that both arsPCom and arsCCom are transcribed (Fig. 3a). Based on this result, it is proposed that arsPCom and arsCCom were transcribed and functional in the presence of arsenate. Moreover, RT-PCR products between arsP and arsB and between arsB and arsC were obtained (Fig. 3a), indicating that arsP and arsB, and similarly arsB and arsC, were cotranscribed. It is therefore concluded that arsP, arsB and arsC belong to the same transcriptional unit (Fig. 1a).
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) for the arsenate-stressed proteome. In addition six proteins were decreased in abundance (2.4–3.9-fold) or disappeared in the arsenate-stressed proteome.
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). To improve the protein identification, a local dataset was established based on the draft genome sequence of strain CNB-1. By using this specific protein dataset, 23 proteins were identified (Table 2). Among those proteins, one protein, ArsPCom (Fig. 3b shows the results from three parallel experiments, and Fig. 3c indicates its PMF), was directly involved in arsenate resistance. Other proteins directly involved in arsenate resistance for strain CNB-1 were not detected on the 2-DE gels. Interestingly, more proteins involved in other cellular processes were significantly influenced in the presence of arsenate; these proteins are described below.
Stress response, protein refolding and ribosomal proteins.
Heat-shock proteins that were newly synthesized or have their abundance increased in the presence of arsenate included DnaJ, DnaK (HSP70) and HSP20, which play an important role in protein refolding under stress conditions. Another protein involved in repair of damaged protein was glutathione S-transferase, which increased its abundance by 2.5-fold. Three ribosomal proteins, S1, L1 and L3, decreased in abundance, suggesting that the protein synthesis process was affected in the presence of arsenate.
Carbohydrate metabolism and energy production.
Other proteins identified included enzymes involved in carbohydrate metabolism and the glyoxylate cycle. In the presence of arsenate, enolase increased by 4.8-fold. Enolase is a glycolytic enzyme catalysing the formation of phosphoenolpyruvate from 2-phosphoglycerate, the second of two high-energy intermediates that generate ATP in glycolysis. It was also found that a subunit of ubiquinol-cytochrome c reductase, which is involved in oxidative phosphorylation and leads to ATP generation, decreased by 2.8-fold (Table 2). Maintenance of energy (ATP) in a cell is critical for cell growth and proliferation; this study found that the enzymes involved in substrate-level phosphorylation increased but oxidative phosphorylation decreased when Comamonas strain CNB-1 grew in the presence of arsenate. Malate synthase G, an important characteristic enzyme of the glyoxylate cycle, was newly synthesized in the presence of arsenate. It is an enzyme catalysing the reversible condensation of glyoxylate with acetyl-CoA and water to form malate and CoA. The expression of malate synthase G enabled the cell to use acetyl-CoA to generate increased levels of TCA cycle intermediates available for cells.
Membrane transport system.
There were three induced proteins that were components of the membrane transporter system. They were the outer-membrane lipoprotein of the RND efflux system, the periplasmic phosphate-binding protein of the phosphate ABC transporter, and the extracellular solute-binding protein, respectively, indicating that the occurrence of arsenate in culture broth had an impact on the membrane transporter system.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; San Francisco et al., 1990; Wu & Rosen, 1991; Chen & Rosen 1997; Wang et al., 2006; Muller et al., 2007), the unique property of this ars operon from pCNB1 lies in that the ars operon has a total of four genes, namely arsRCom, arsPCom, arsCCom and arsBCom, and that arsCCom and arsPCom encode two arsenate reductases. Genetic complementation with plasmids harbouring either arsCCom or arsPCom in arsC-negative E. coli conferred arsenate resistance to the host cells, and RT-PCR revealed that both arsCCom and arsPCom were transcribed in the presence of arsenate. Although only ArsPCom was detected from the proteome of cells exposed to arsenate, expression of arsCCom could not be excluded. In fact, a careful comparison of the proteomic gel images obtained from cells grown with and without arsenate revealed two spots that specifically appeared in the presence of arsenate; the pI/Mr of the two spots were determined to be 6.02/15 000 and 5.71/13 000, which were close to the theoretical values (6.04/13 282 Da) of ArsCCom. But identification of the two spots with MALDI-TOF MS failed. ArsCCom is homologous and phylogenetically related to the family I arsenate reductases that use glutaredoxin as electron donor and were previously identified in bacteria (Chen et al., 1986; Ji & Silver, 1992; Rosen, 2002), and ArsPCom is phylogenetically related to the family II arsenate reductases that use thioredoxin as electron donor. Very recently, two genes, arsCa (annotated as arsenate reductase) and arsCb (annotated as arsenate efflux), that are located on the Herminiimonas arsenicoxydans genome and homologous to arsCCom and arsPCom, respectively, have been reported (Muller et al., 2007), but they were not physiologically identified. Our experimental results clearly indicated that both ArsPCom and ArsCCom restored arsenate resistance of ArsC-negative E. coli, and they also showed significant difference in the level of arsenate resistance. ArsCCom enabled the ArsC-negative E. coli to resist much higher levels of arsenate. More biochemical work is needed for a full characterization of the two arsenate reductases encoded by arsPCom and arsCCom.
In addition to the synthesis of arsenate-resistance proteins, other cellular responses to exposure of arsenate were also observed in Comamonas strain CNB-1 and other prokaryote strains with the proteomic tools in this and previous studies (Carapito et al., 2006; Parvatiyar et al., 2005; Baker-Austin et al., 2007) (a diagram showing all the cellular processes influenced by arsenate in strain CNB-1 is shown in supplementary Fig. S3, available with the online version of this paper). In the acidophilic Ferroplasma acidarmanus strain Fer1, synthesis of heat-shock proteins HSP60 and HSP70, which are involved in protein refolding, was enhanced when cells were exposed to arsenite [As(III)] (Baker-Austin et al., 2007). In P. aeruginosa, exposure to arsenite resulted in an oxidative-stress-like response (Parvatiyar et al., 2005). This current study has shown for the first time the cellular responses to arsenate [As(V)] of Comamonas strain CNB-1. The global response to arsenate in strain CNB-1 included the enhanced synthesis of proteins associated with arsenate resistance and detoxification (ArsPCom and GST), phosphate transport (PstS), heat-shock response (DnaJ/DnaK/HSP20), and energy generation and carbon metabolism (malate synthase and enolase). Functions of these proteins in arsenate resistance by Comamonas CNB-1 are illustrated in Fig. S3. Different from the response to arsenite in P. aeruginosa (Parvatiyar et al., 2005), significant changes of proteins involved in oxidative stress-like response were not observed in Comamonas strain CNB-1 when exposed to arsenate. The DnaK/DnaJ chaperone system is essential for the recovery of stress-induced protein aggregates (Kedzierska, 2005); thus it was deduced that exposure to arsenate would caused significant protein misfolding in cells. The increased DnaK/DnaJ abundance would stimulate the recovery of the misfolded protein due to the arsenate/arsenite binding.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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Received 10 July 2007;
revised 13 August 2007;
accepted 15 August 2007.
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, WC3110; 
, WC3110/pSE380-arsCCom; 
, WC3110/pSE380-arsPCom. Data were from three parallel experiments and standard deviations are indicated.