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Journal of Bacteriology, December 2007, p. 8807-8817, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00960-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiple Superoxide Dismutases in Agrobacterium tumefaciens: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis{triangledown}

Panatda Saenkham,1 Warawan Eiamphungporn,1,{dagger} Stephen K. Farrand,2 Paiboon Vattanaviboon,3 and Skorn Mongkolsuk1,3,4*

Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand,3 Department of Biotechnology,1 Centre of Emerging Bacterial Infection, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,4 Department of Microbiology, University of Illinois at Urbana-Campaign, Urbana, Illinois 618012

Received 18 June 2007/ Accepted 25 September 2007


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ABSTRACT
 
Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.


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INTRODUCTION
 
Agrobacterium tumefaciens is a gram-negative aerobic bacterium commonly found in rhizospheres. The bacterium is the causative agent of crown gall disease on a variety of dicotyledonous plants. During infection, the bacterium transfers part of its Ti plasmid, the T-DNA, into the plant cell, resulting in tumor formation (12).

Superoxide anion (O2·–) is a toxic by-product of aerobic metabolism mainly produced by a partial reduction of oxygen molecules by electrons leaking from the respiratory chain. It can also be generated by auto-oxidation of redox-cycling compounds such as menadione (MD), plumbargin, and the herbicide paraquat and by the host defense response against invading microorganisms. The toxicity of a superoxide anion arises from its ability to oxidize and leach iron from iron-sulfur cluster (4Fe-4S)-containing proteins, thereby inactivating enzymes such as aconitase, 6-phosphogluconate dehydratase, and fumarases (28). In addition, the released iron could participate in the Fenton reaction, leading to increased production of hydroxyl radicals, which can cause damage to many macromolecules including DNA (28). Superoxide anions also can be dismutated to H2O2, which itself is reactive to macromolecules and can participate in the Fenton reaction.

Bacteria have evolved an impressive array of antioxidant defenses to deal with superoxide anion. Superoxide dismutase (Sod; EC 1.15.1.1) is a metalloenzyme catalyzing the dismutation of superoxide anions to form H2O2, a substrate of catalases, and molecular oxygen. The enzymes can be divided according to their metal cofactors into four classes, namely, the manganese (MnSod), the iron (FeSod), the copper-zinc (Cu/ZnSod), and the nickel (NiSod) (19). Several gram-negative bacteria produce multiple Sod enzymes which differ in terms of metal cofactor, subcellular location, and regulation of gene expression. Escherichia coli has three Sods: MnSod (encoded by sodA), FeSod (encoded by sodB), and Cu/ZnSod (encoded by sodC). The first two enzymes are cytoplasmic, while the latter locates to the periplasmic space (19). FeSod is constitutively expressed, while MnSod is produced in the presence of oxygen, upon response to accumulation of intracellular superoxide anion, or upon a change in bacterial growth phase. Consistent with this pattern of regulation, sodA is a member of the SoxRS regulon (19, 34). The contribution of Sods to bacterial virulence has been investigated for several animal pathogens. In most cases, inactivation of genes encoding Sods impairs bacterial survival in tested models (32). Although increased production of reactive oxygen species (ROS) is also a common feature in plant defense systems, the roles of Sod as a virulence factor in phytopathogenic bacteria are still unclear, due to the limited number of investigations and variations observed in different plant-microbe interactions. In Xanthomonas campestris, the expression of an MnSod is induced during bacterial invasion into the host plant (42). However, the role of this enzyme in pathogenicity is not clear. Deletion of sodA in Erwinia chrysanthemi reduced the size of necrotic lesions on African violets, while the ability to infect potato was unaffected (40). However, Sod has a key protective role in the symbiotic process of Sinorhizobium meliloti (41). In this communication, we report the characterization of three Sod isozymes in A. tumefaciens. Mutants lacking one or all three of the Sod isozymes showed moderate to severe defects in their abilities to induce tumors on tobacco leaves.

(Parts of this work are from the dissertation of P.S. submitted for a Ph.D. degree from Mahidol University.)


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MATERIALS AND METHODS
 
Bacterial growth conditions. Agrobacterium tumefaciens NTL4 (a pTiC58-cured derivative of C58 {Delta}tetC58) (35) and the mutant strains were grown aerobically in Luria-Bertani (LB) medium at 30°C with continuous shaking at 150 rpm. To ensure comparable growth states, overnight cultures were inoculated into fresh LB medium to give an optical density at 600 nm (OD600) of 0.1. Exponential-phase (OD600 of 0.6 after 4 h of growth) and stationary-phase (OD600 of about 5 after 30 h of growth) cells were used as indicated in all experiments. The oxidant-induced experiments were performed by adding either 250 µM H2O2, 250 µM tert-butyl hydroperoxide, or 200 µM MD to exponential-phase cultures. These cultures were grown for an additional 15 min for Northern analysis and 30 min for enzymatic assays.

Molecular biology techniques. General molecular genetics techniques, including genomic DNA preparation, plasmid preparation, restriction endonuclease digestion, ligation, transformation in E. coli, agarose gel electrophoresis, and Southern and Northern blot analyses were performed using standard protocols (38). Plasmids were purified for DNA sequencing using Qiagen Miniprep kits (Qiagen, Germany). Routinely, A. tumefaciens was transformed by electroporation as previously described (35).

Primer extension. Primer extension experiments for sodBI were performed using 10 µg of total RNA, 200 U of Superscript III reverse transcriptase (Invitrogen), and 32P-labeled oligonucleotide primer BT1269 (Table 1). The sizes of the extension products were determined on sequencing gels (38) by comparison to dideoxy sequencing ladders generated using a PCR sequencing kit with labeled pUC/M13 forward primer and pGEM-3Zf(+) as the template (Applied Biosystems). Primer extension of sodBII was performed using the same protocol described for sodBI except the total RNA samples were extracted from uninduced and MD-induced cultures and the 32P-labeled oligonucleotide primer BT817 was used.


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  		TABLE 1. Bacterial strains and primers used in this study

Construction of A. tumefaciens sodBI, sodBII, and sodBIII mutants and full-length genes. A. tumefaciens sodBI, sodBII, and sodBIII mutants were constructed by insertion inactivation using suicide plasmid pKNOCK (1). DNA fragments (~200 bp) of the target gene were amplified in a PCR using NTL4 genomic DNA as a template. The specific primers for sodBI (BT534 and BT535), sodBII (BT640 and BT641), and sodBIII (BT644 and BT645) were designed based on the nucleotide sequences corresponding to putative open reading frames (ORFs) Atu0876 (accession no. AAL41890), Atu4583 (accession no. AAL45377), and Atu4726 (accession no. AAL45520), respectively (51). The PCR products were ligated into pGEM-T Easy (Promega) prior to the subcloning of the EcoRI fragments into the pKNOCK vector. The recombinant plasmids were transferred into NTL4 by conjugation (1). The putative mutants were screened by PCR using two primers flanking the insertion sites and confirmed by Southern blot analysis. The full-length genes were amplified from NTL4 genomic DNA with primers BT638 and BT639 for sodBI, BT711 and BT729 for sodBII, and BT709 and BT710 for sodBIII by use of Pfu polymerase (Promega). The PCR products were cloned into broad-host-range plasmid pBBR1MSC4 (31) and digested with EcoRV, giving pSodBI, pSodBII, and pSodBIII. The cloned sods were all sequenced.

sodBI, sodBII, and sodBIII promoter-lacZ fusion. Promoter regions of sodBI and sodBIII were fused to suicide promoterless plasmid vector pVIK112 (30). The sodBI fragment was amplified using BT534 and BT535 primers and cloned into pGEM-T Easy (Promega). The EcoRI-SpeI blunt fragment was subsequently cloned into pVIK112 digested with EcoRI and SmaI. The resultant plasmid was introduced into A. tumefaciens NTL4. Homologous recombination between the sodBI fragment on the nonreplicating plasmid and on the chromosome generated a sodBI::lacZ fusion. The strain containing this fusion was designated PS1. Strain PS2, containing a sodBIII::lacZ fusion, was obtained using the same protocol, but the sodBIII fragment was amplified with BT644 and BT645 primers prior to cloning into pVIK112. The sodBII promoter-lacZ gene fusion was constructed in a low-copy-number promoter-probed vector, pUFR027lacZ (36). The sodBII promoter region was amplified using BT640 and BT793 primers and A. tumefaciens NTL4 genomic DNA as a template. The 680-bp PCR product was first cloned into pGEM-T Easy (Promega) before the EcoRI fragment was subcloned into pUFR027lacZ digested with the same enzyme to generate pPsodBII.

SoxR purification. A. tumefaciens SoxR was overexpressed in E. coli BL21 by use of pETsoxR (15), which contains full-length soxR in the pETBlue-2 vector (Novagen). Oxidized SoxR was purified over a P-11 phosphocellulose ion-exchange column and was eluted with 0.5 M KCl as previously described (15). The purified A. tumefaciens SoxR showed a UV spectrum that was similar to the spectrum obtained from E. coli oxidized SoxR (25).

Gel mobility shift assay. Gel mobility shift reactions were performed by preparing 3 fmol of labeled probe (32P-labeled 220-bp sodBII promoter fragment amplified using BT1142 and BT817 primers) in 25 µl of reaction buffer [20 mM Tris (pH 7.0), 50 mM KCl, 1 mM EDTA, 5% glycerol, 50 µg ml–1 bovine serum albumin, 5 µg ml–1 calf thymus DNA, 0.5 mg ml–1 poly(dI-dC)]. Various amounts of purified SoxR (15) were added, and the reaction mixture was incubated at 25°C for 15 min. Protein-DNA complexes were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer at 4°C and visualized by exposure to X-ray film.

Determination of oxidant resistance. Levels of resistance to oxidants were determined using a plate sensitivity assay as previously described (15). Serial dilutions of bacterial cultures were made in 50 mM sodium phosphate buffer, pH 7.0, for stationary-phase cells and in LB broth for exponential-phase cells, and 10 µl of each dilution was spotted onto an LB agar plate containing 0.5 mM MD. The plates were incubated at 30°C for 48 h before the results were recorded. The resistance levels were expressed as percent survival, defined as the CFU on plates containing oxidant divided by the CFU on plates without oxidant multiplied by 100.

Resistance towards extracellular superoxide anions was assayed using the xanthine-xanthine oxidase system as described previously (47). Stationary-phase cells were treated with 4.3 mM xanthine, 1 U ml–1 xanthine oxidase, and 1 U Xanthomonas KatA monofunctional catalase (11) for 30, 60, and 90 min at 30°C. Bacterial cells surviving the treatment were scored using standard viable cell counts on LB agar plates.

Sod activity gels. To visualize Sod activity, 50 µg proteins from crude samples were separated by nondenaturing electrophoresis performed using a 10% polyacrylamide gel (pH 8.5) with a 5% stacking gel (pH 6.9) followed by staining with nitroblue tetrazolium-riboflavin photochemical stain as described by Beauchamp and Fridovich (5). In the enzyme inhibition tests, the gel was incubated with 10 mM H2O2, sodium azide, or potassium cyanide for 45 min prior to enzyme activity staining. Comparison of Sod activities and the percents inhibition of Sod activities were estimated using densitometry analysis.

Alkaline phosphatase assays. Alkaline phosphatase assays were performed according to the method described by Garen and Levinthal (21), in which the activity was determined by monitoring an increase in OD410 resulting from the hydrolysis of p-nitrophynyl phosphate to p-nitrophenol. One unit is defined as the amount of enzyme capable of releasing 1 micromole of p-nitrophenol per min at 25°C and pH 8.0.

Virulence assay. Virulence assays were performed as described previously (44) with some modifications. In brief, Nicotiana tabacum leaf explants and the wild type or sod mutants of A. tumefaciens were cocultivated for 48 h on hormone-free Murashige and Skoog (MS) medium containing 60 µg ml–1 acetosyringone. The infected leaf discs were then transferred to fresh MS medium containing 200 µg ml–1 Timentin (ticarcillin disodium-clavulanate potassium; GlaxoSmithKline) to inhibit bacterial growth. After 14 days of incubation in the dark, the numbers of tumors on each leaf explant were scored from 0 to 5 by comparison to a standard, with 0 representing tumors induced by NTL4 without a Ti plasmid (pCMA1) and 5 representing tumors induced by NTL4 bearing the Ti plasmid pCMA1. At least 20 leaf explants were used for each strain tested, and each experiment was repeated three times.


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RESULTS AND DISCUSSION
 
A. tumefaciens has three FeSods. A BLASTP (3) search of the annotated genome sequence of A. tumefaciens C58 (51) with E. coli Sod amino acid sequences available at http://www.ncbi.nlm.nih.gov/ identified three ORFs, namely, Atu0876 (SodBI), Atu4583 (SodBII), and Atu4726 (SodBIII), with high sequence homology. sodBI (formerly sod1 [49]) is located on the circular chromosome, while sodBII and sodBIII (formerly sod2 [49]) are on the linear chromosome. SodBI, SodBII, and SodBIII share 43.5%, 42.5%, and 36.8% identities, respectively, to E. coli iron-containing Sod (SodB). Percentage identities among A. tumefaciens SodB are 46.7% (SodBI and SodBII), 35.5% (SodBI and SodBIII), and 35.2% (SodBII and SodBIII). The analysis of the primary structure of these putative Sods by use of the BLAST program (3) showed that they belong to the Fe/MnSod family. Multiple alignments of these ORFs with known Fe- and Mn-containing Sods by use of CLUSTAL W (46) revealed that all amino acid residues involved in metal binding and the enzyme active sites are conserved in the three A. tumefaciens Sods (Fig. 1). The presence of conserved key amino acid residues described in the work of Santos et al. (39) also suggests that sodBI, sodBII, and sodBIII encode FeSod isozymes.


Figure 1
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  		FIG. 1. Multiple alignments of Sods. Multiple alignments of the deduced amino acid sequences of Sods from A. tumefaciens (Atu) (51) with FeSod (SodB) and MnSod (SodA) from Pseudomonas aeruginosa (Pae) (24) and from E. coli (Eco) (10, 43).

The inhibition of Sod activity by H2O2, cyanide, and azide has been used to differentiate metal cofactors of various Sods (19). Sod activity gels and various inhibition tests were performed to establish the metal cofactor of the three putative A. tumefaciens Sods by use of crude extracts prepared from a triple sod null mutant (sodBI sodBII sodBIII mutant) harboring either pSodBI, pSodBII, or pSodBIII (Fig. 2A). The results demonstrate that all three putative genes encode enzymes with Sod activity, as shown by a positive reaction in Sod activity gels. Furthermore, treating the Sod activity gels with 10 mM H2O2 inhibited all A. tumefaciens Sod isozymes, while these enzyme activities were resistant to treatment with cyanide and azide (Table 2). We conclude on the basis of inhibition tests and sequence analysis that SodBI, SodBII, and SodBIII are FeSods. In this regard, A. tumefaciens differs from most other bacteria which express cytoplasmic Sods, either Fe or Mn or both Fe and Mn Sods. Under superoxide and peroxide stress conditions, bacteria generally chelate free iron and reduce iron uptake to minimize the participation of free iron in various free radical-generating reactions. The physiological advantage to A. tumefaciens of having only FeSods is not clear. It could be that the FeSod is functionally better for the bacterium in the soil, rhizosphere, or plant wound site environment.


Figure 2
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  		FIG. 2. Multiple Sods in A. tumefaciens. Sod activity gels of A. tumefaciens strains. Equal amounts of protein (50 µg) from crude extracts of NTL4 were loaded and separated on a native polyacrylamide gel. (A) sodB null mutant harboring plasmid for high expression of sodBI (pSodBI), sodBII (pSodBII), and sodBIII (pSodBIII). (B) A. tumefaciens NTL4 cultivated to different time points (4 h, 8 h, 24 h, and 30 h) and the exponential-phase culture grown under uninduced (UN) and MD-induced conditions. The right panel shows Sod activity from an NTL4 stationary-phase culture and a sodBIII mutant. (C) Sod activity from the exponential growth of mutant strains challenged with oxidants. BH, tert-butyl hydroperoxide. Arrowheads represent the positions of SodBI, SodBII, and SodBIII activity as indicated.


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  		TABLE 2. The H2O2, KCN, and NaN3 inhibitions of SodBI, SodBII, and SodBIII activities

Subcellular localization of SodBI, SodBII, and SodBIII. The amino acid sequences of SodBI and SodBII lack detectable signal peptide sequences and most likely are cytoplasmic enzymes. By contrast, TatP analysis (6) (available at http://www.cbs.dtu.dk/services/TatP/) of the SodBIII sequence showed an extended N terminus and a twin-arginine signal sequence with a potential cleavage site between positions 29 and 30 (AQA-AL), suggesting that the enzyme is localized to the periplasm. We constructed translational gene fusions downstream of the sodBIII coding region for a signal sequence with either phoA (giving pSodIII-phoA) or lacZ (giving pSodIII-lacZ) in pBBR1MSC4 (31) to confirm the location of the enzyme. The alkaline phosphatase activity specified by NTL4 bearing pSodBIII-phoA was 0.35 U mg–1 protein compared to 0.05 U mg–1 protein in NTL4 harboring pPhoA, a clone of phoA with a promoter but no signal sequence. No β-galactosidase activity was detected in strains harboring pSodBIII-lacZ. These results favor the periplasmic localization for SodBIII. This raises an important question how iron gets inserted into the periplasmic SodBIII. Examination of the SodBIII leader sequence shows twin-arginine (RR) residues in the leader sequence, suggesting that the TAT system could be involved in the transportation of the protein. It is likely that newly synthesized SodBIII is folded with metal insertion in the cytoplasm before being transported to periplasm by the TAT system.

Generally, periplasmic Sods have multiple physiological roles. First, they provide the first line of defense against externally generated superoxide anions. Second, they protect periplasmic enzymes from inadvertently generated superoxide anions from the electron transport chain. In most bacteria, the predominant periplasmic Sods have Cu/Zn metal cofactors (29). Only a few bacteria have been reported to produce periplasmic MnSod (33) or FeSod (18). The BLASTP search of the A. tumefaciens genome failed to identify a homolog of Cu/ZnSod. Thus, A. tumefaciens has evolved and maintained a FeSod in the periplasm.

The three levels of the three Sods differed during different growth phases as well as in their responses to oxidants. Analysis of total Sod activity in crude extracts taken from an A. tumefaciens culture grown in rich medium (LB) at various growth phases by use of Sod activity gels revealed a single Sod activity band (F-Sod) detected during exponential phase, while an additional slower-migrating Sod (S-Sod) activity band was observed during the stationary phase of growth (Fig. 2B). Challenging the exponential cultures with sublethal concentrations of MD, a superoxide generator, caused a substantial increase in the level of F-Sod activity (Fig. 2B). Total Sod activity was increased from 0.47 ± 0.05 U mg–1 protein in uninduced cells to 1.40 ± 0.10 U mg–1 protein in MD-induced cells. These observations suggest that the expression levels of the three sods differ with respect to growth phase and response to oxidants.

To identify the ORFs that encode the Sod activities observed with the activity gels, strains deleted for sodBI, sodBII, or sodBIII were constructed. The results show that the F-Sod activity band was diminished for a sodBI mutant (Fig. 2C), while the S-Sod activity band disappeared for a sodBIII mutant (Fig. 2B). The identities of these Sods were confirmed by expression of either pSodBI or pSodBIII in the respective mutants; in each case, the complementing plasmids restored the appropriate activity band (data not shown). The sodBII mutant did not show significant alterations in F-Sod or S-Sod activity bands. However, the expression of pSodBII in a mutant lacking three sods produced an activity band that comigrated with SodBI at the F-Sod activity band position (data not shown).

sodBI is highly expressed throughout all growth phases. As assessed by Northern blot analyses, sodBI was transcribed as a monocistronic mRNA and expressed at a high level, as judged by the size and intensity of the Northern blot-hybridized signals (Fig. 3A). Treatments with oxidants did not affect levels of sodBI expression. Primer extension was performed to locate the sodBI promoter. Two bands of primer extension products were detected in RNA samples prepared from exponential-phase culture. A major band of 110 bp corresponds to a transcription initiation site at a T residue (+1P1) 49 nucleotides upstream of the translation initiation codon (Fig. 3B). The sodBI promoter had sequence motifs TTGCCC and TATGGT at the –35 and –10 regions, respectively, separated by 17 bp. The sodBI promoter resembles typical strong promoters of A. tumefaciens genes (13, 15). The second primer extension products corresponded to the 5' end of sodBI, located at the G residue (+1P2) 7 nucleotides downstream of +1P1. This gave putative –35 (CAAACA) and –10 (TCCGCT) promoter regions which were atypical of A. tumefaciens promoters, suggesting that the observed second primer extension product was a result of premature termination of the reverse transcription in the reaction. The analysis of sodBI in vivo promoter activity was performed for strain PS1 containing a chromosomal sodBI promoter region fused to lacZ. The sodBI promoter activity was maintained at high levels throughout all growth phases (0.28 U mg–1 protein at exponential phase and 0.48 U mg–1 protein at stationary phase). Treatment of the cultures with oxidants failed to induce β-galactosidase activity to higher levels, confirming the Northern analysis results (data not shown). sodBI is expressed at high levels throughout all growth phases, and the gene does not respond to oxidants.


Figure 3
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  		FIG. 3. Transcriptional analysis of the sodBI gene in A. tumefaciens. (A) Northern analyses of sodBI expression in A. tumefaciens NTL4 under uninduced (UN) conditions and induction with H2O2, MD, and tert-butyl hydroperoxide (BH). 16S rRNA levels were used as loading controls and are shown underneath the autoradiograph of the Northern blot. (B) Primer extension showing the transcriptional start site (+1) of sodBI. Arrowhead indicates +1 nucleotide. C, T, A, and G are sequence ladders of pGEM-3Zf (+) from the pUC/M13 forward primer (Applied Biosystems), and the numbers represent the sizes in base pairs of the sequencing products. The arrowheads and +1P1 and +1P2 indicate the sodBI transcription start sites. The –35 and –10 promoter regions of P1 and P2 are underlined and in gray letters, respectively. The translation codon (ATG) and the putative ribosome binding site of sodBI are in boldface and italics, respectively.

sodBII is a member of the soxR regulon. When an exponential culture of the sodBI mutant was treated with MD, increased Sod activity was detected in a Sod activity gel assay (Fig. 2C). The SodBII activity band was barely visible under normal physiological conditions during all phases of growth (Fig. 2C). In a sodBI sodBIII double mutant, treatments of exponentially growing cultures with MD but not other oxidants such as H2O2 and organic hydroperoxides strongly enhanced levels of SodBII (Fig. 2C). Northern analysis of sodBII expression showed a high level of a hybridized monocistronic mRNA of approximately 0.7 kb in size only in RNA samples from MD-induced cells (Fig. 4A). Treatment of the cultures with other oxidants did not affect the expression of sodBII. The expression profile of sodBII is similar to those observed for other SoxR-regulated genes in A. tumefaciens (15). Hence, Northern analysis was repeated using RNA samples prepared from the uninduced and oxidant-induced soxR mutant PW01 (16). The MD-induced expression of sodBII was abolished in this mutant (Fig. 4A). This result indicates that the induction of sodBII expression with MD is dependent on SoxR. The results also suggest that the activation of sodBII expression is responsible for the increase in total Sod activity observed for MD-induced cells.


Figure 4
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  		FIG. 4. Expression analysis of sodBII in A. tumefaciens. (A) Northern analyses of sodBII expression in A. tumefaciens NTL4 and a soxR mutant (PW01) under uninduced (UN) conditions and induction with H2O2, MD, and tert-butyl hydroperoxide (BH). 16S rRNA levels were used as loading controls and are shown underneath the autoradiograph of the Northern blot. (B) Primer extension showing the transcriptional start site (+1) of sodBII. Arrowhead indicates +1 nucleotide. G, A, T, and C are sequence ladders of pGEM-3Zf(+) from the pUC/M13 forward primer (Applied Biosystems), and the numbers represent the sizes of the sequencing products in base pairs. –10 and –35 regions are boxed. The translation codon (ATG) of sodBII is in boldface. The putative SoxR binding site is shaded. (C) Gel mobility shift assay of the sodBII promoter fragment with increasing amounts of purified SoxR protein (0 to 100 ng). UP and HD indicate the binding reaction using 80 ng SoxR containing an additional 1 µg of unlabeled sodBII promoter and 1 µg of heterologous DNA (pUC18 plasmid), respectively. F and B indicate free and bound probes, respectively.

Next, the location of the sodBII promoter was determined using primer extension mapping performed on total RNA extracted from uninduced and MD-induced cultures. A 112-bp major primer extension product, corresponding to the sodBII transcript start site (+1) located at an A residue 60 bp upstream of the AUG translation start codon, was detected in an MD-induced RNA sample. This product was barely detectable in the RNA sample from uninduced cells (Fig. 4B). This result confirms that the MD-dependent increase in sodBII expression is due to increased transcription from the sodBII promoter. Examination of the sequence upstream of the +1 site revealed the presence of two sequence motifs, TTGACC (–39 to –34) and TAGAAG (–14 to –9), corresponding to the –35 and –10 promoter regions, respectively (Fig. 4B). These promoter motifs were separated by 19 bp, which is typical for SoxR-regulated promoters (26). Furthermore, a search for a SoxR box near the promoter region identified a putative SoxR box with high sequence homology (12 out of 18 bp matched) to a consensus sequence for the E. coli SoxR binding site (37) located between the –10 and –35 regions (Fig. 4B).

In A. tumefaciens, SoxR binds to its recognition box and directly regulates the expression of genes in its regulon (15). We tested the ability of purified oxidized SoxR protein to bind to the sodBII promoter by use of a DNA mobility shift assay. Oxidized SoxR specifically bound to the sodBII promoter (Fig. 4C). The binding specificity was shown by the ability of the unlabeled soxR promoter fragment but not an excess of heterologous DNA to compete with SoxR binding to the labeled promoter fragment (Fig. 4C).

sodBII expression was monitored throughout the growth phase by use of the sodBII::lacZ fusion in pPsodBII. The results suggest that sodBII is expressed at low levels throughout all phases of growth (data not shown). MD treatment caused an 11-fold increase in β-galactosidase activity (from 0.79 ± 0.03 to 9.08 ± 0.26 U mg–1 protein) in wild-type strain NTL4 harboring pPsodBII. However, induction by MD was abolished in the soxR mutant PW01/pPsodBII (uninduced, 0.81 ± 0.05; MD induced, 0.75 ± 0.05). MD inducibility could be restored to PW01/pPsodBII by expression of soxR from a plasmid (uninduced, 0.87 ± 0.06; MD induced, 10.97 ± 0.24). These results are in good agreement with the data obtained from Northern analysis. We speculated that upon exposure to superoxide stress, A. tumefaciens SoxR is being oxidized and that the oxidized form of SoxR binds to the SoxR box located between the –10 and –35 regions of the sodBII promoter, where it activates the transcription of the gene (14).

Genes in the SoxR regulon can be repressed or activated depending on whether reduced or oxidized SoxR is bound. The data presented here suggest that oxidized SoxR activates the expression of sodBII. For E. coli, SoxR has been shown to exist in a reduced form in uninduced cells (22). Thus, we extended the investigation and determined the role of reduced SoxR on the sodBII promoter by use of in vivo sodBII promoter analysis. Measurements of the basal levels of β-galactosidase activities specified by pPsodBII in NTL4 (0.79 ± 0.03 U mg–1 protein), PW01 (0.81 ± 0.04 U mg–1 protein), and PW01/pSoxR (0.87 ± 0.06 U mg–1 protein) showed no significant differences. These results suggest that reduced SoxR has no direct effects on the sodBII promoter. This observation contrasts with previous observations for A. tumefaciens, where in vivo-reduced SoxR repressed the expression of atu5152, a member of SoxR regulon, and itself (15). Nonetheless, this observation fits well with the SoxR paradigm that reduced SoxR either represses or has no effect on the expression of genes while oxidized SoxR only activates the expression of genes in its regulon.

sodBIII is expressed during stationary phase. In initial experiments, we could not reliably detect sodBIII mRNA in Northern blot experiments, suggesting that the gene is expressed at a low level. The expression of sodBIII was investigated for A. tumefaciens strain PS2 containing the sodBIII promoter region fused to lacZ. β-Galactosidase activity in PS2 culture was assayed throughout the growth phases. As shown in Fig. 5, sodBIII exhibited showed a sixfold increase in β-galactosidase activity, from 0.006 U mg–1 protein at exponential phase to 0.038 U mg–1 protein at stationary phase (30 h). This pattern of sodBIII expression is consistent with the observations from activity gels, which showed that SodBIII levels increased during the stationary phase of growth observed in a Sod activity gel assay (Fig. 2B).


Figure 5
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  		FIG. 5. Transcriptional analysis of the sodBIII gene in A. tumefaciens. β-Galactosidase activity levels in PS2 containing sodBIII promoter lacZ fusions were plotted against OD600 values at various time points.

A similar pattern of stationary-phase expression for periplasmic Cu/ZnSods has been reported for other bacteria (29). The sodBIII expression pattern of sodBIII resembles many of the RpoS-regulated genes found in other gram-negative bacteria. However, A. tumefaciens lacks an RpoS homologue, and other regulators of stationary-phase gene expression have not been identified.

Functional analysis of sods. In many bacteria, the physiological function of sod is to alleviate superoxide stress by lowering the steady-state concentration of superoxide anions. Results presented here show differential expression patterns of the three sods as well as differing cellular localizations for these enzymes. The physiological roles of the three sods in A. tumefaciens are not clear. Hence, insertion inactivation mutants in individual sod gene, combinations of individual sod mutations, and a triple sod null mutant were constructed to evaluate the physiological roles of the three isozymes. The growth rates of the individual sod mutants under aerobic conditions in either rich or minimal medium were not significantly different from that for NTL4, the wild type (data not shown). We then tested each mutant for levels of resistance against lethal concentrations of superoxide generators in exponential- and stationary-phase cells. At exponential phase, the inactivation of sodBI rendered A. tumefaciens 70-fold more sensitive to killing by MD compared to what was seen for NTL4 (Fig. 6A). The phenotype could be complemented by expression of sodBI from pSodBI. The expression patterns and the sodBI mutant phenotype suggest that the gene has a housekeeping role in ensuring that the levels of intracellular superoxide anions are kept at low levels throughout the different phases of growth. The inactivation of sodBI caused the highest reduction in total Sod activity under uninduced conditions. Hence, the inactivation of the gene results in the most pronounced decrease in the overall resistance to superoxide conferred by the three sods.


Figure 6
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  		FIG. 6. Phenotypes of various A. tumefaciens sod mutant strains. (A) The survivals of A. tumefaciens strains on a plate containing 0.5 mM MD were determined for NTL4 and for sodBI, sodBII, sodBIII, sodBI sodBII, sodBI sodBIII, sodBII sodBIII, and sodBI sodBII sodBIII (null) mutant strains. (B) A. tumefaciens NTL4 and sodBI, sodBII, sodBIII, and sodBI sodBII sodBIII (null) mutant strains containing the Ti plasmid (pCMA1) were tested for their abilities to induce tumor formation on pieces of tobacco leaves as described in Materials and Methods. Control means leaf explants infected with NTL4 without pCMA1. (C) The sodBI sodBII sodBIII mutants (null) harboring pSodBI, pSodBII, pSodBIII, and pSodBIII{Delta}29 as indicated were tested for tumorigenesis as described for panel B. Photos were taken after incubation for 14 days.

SodBIII is a periplasmic enzyme that is induced during stationary phase. The inactivation of sodBIII alone had no effect on the MD resistance levels during exponential growth. The cellular location of SodBIII suggests that it could have a physiological role in protecting the bacteria from externally generated superoxide anions. To test this hypothesis, a sodBIII mutant was treated with xanthine-xanthine oxidase in a killing assay (47) in the presence of bacterial monofunctional catalase, and no difference in the survival rates for the mutant and the parental strain was observed (data not shown). These data suggest that sodBIII plays a minor role in protecting the bacteria from superoxide stress during exponential phase. Moreover, the remaining Sod activities could compensate for the lack of SodBIII activity. The inactivation of sodBII resulted in a small decrease in MD resistance levels during exponential phase (Fig. 6A), implying that the inducible sodBII plays a minor protective role against MD toxicity in uninduced growth conditions.

The presence of multiple Sod activities prevents a clear analysis of the role of each sod in protecting the bacteria from superoxide stress. Hence, combinations of double and triple sod null mutants were constructed. All double mutants had an additional decrease in MD resistance levels compared to what was seen for the single mutants. The sodBII sodBIII mutant was the least affected (3-fold) compared to the sodBI sodBIII (20-fold) and the sodBI sodBII (100-fold) mutants. Furthermore, the MD resistance level was lower in a sod null mutant than in any of the single and double sod mutants (Fig. 6A). These findings confirmed that all three sods contribute to various degrees to overall resistance to MD during exponential phase. The contribution of each of the sods to MD resistance appears to correlate with their expression patterns and levels. The inactivation of highly expressed sodBI had the most effect, and that of weakly expressed sodBIII the least effect, on levels of resistance to MD. Additional experiments were done to determine the resistance levels of the mutant strains under acidic pH (5.5) and low-phosphate concentration growth conditions, which mimic the conditions that bacteria encounter during infection of plant wound sites. MD plate sensitivity tests were performed using AB minimal medium, pH 5.5 (9). The results show that all sodB mutants had MD resistance profiles similar to those observed using a rich medium (data not shown). This suggests that sodBs probably have an important role in the protection of A. tumefaciens from superoxide anion during the infection of plant tissues.

In bacteria, stationary-phase cells are generally more resistant to stresses, including superoxide stress, than are exponential-phase cells (7, 17, 48). The mechanisms responsible for this phenotype often are not directly correlated to levels of stress detoxification and protective enzymes, but other factors, such as nonspecific DNA binding proteins and membrane modifications, could be involved (2, 23). Here, unlike what is seen for other bacteria, the resistance of A. tumefaciens to superoxide stresses slightly decreased (fivefold) during the stationary phase of growth, despite a sharp increase in the levels of SodBIII and the maintenance of basal levels of SodBI and SodBII (Fig. 5 and 6A). The initial analysis of individual sod mutants did not show stationary-phase-specific alterations in MD resistance levels. The decreases in the MD resistance levels during stationary phase in single sod mutants were proportional to the decreases in resistance levels observed at the exponential phase. Similarly, the sodBII sodBIII double mutant did not show altered stationary-phase resistance to MD compared to what was seen for the other two single mutants. This suggests that in the double mutant the remaining SodBI can compensate for the absence of the two missing Sod activities during stationary phase. Also, sodBII appears to play a minor role in the basal levels of stationary phase to MD. Nonetheless, in the absence of sodBI and the stationary-phase-specific expression of sodBIII, a sodBI sodBIII double mutant shows an additional fivefold decrease in resistance to MD. These observations suggest that sodBI alone could compensate for the lack of sodBII and sodBIII. We conclude that the highly expressed sodBI is responsible for a large percentage of the total MD stationary-phase resistance. Moreover, sodBIII also plays a significant role in the process, while the expression of sodBII alone is not sufficient for maintaining stationary-phase resistance levels. The sod null mutants show an additional 10-fold decrease in stationary-phase MD resistance levels compared to double sod mutants, indicating that sodBI, sodBII, and sodBIII all contribute to various degrees to the overall resistance level.

We assessed the ability of an individual sod gene in an expression vector to complement the MD-hypersensitive phenotype of the sod null mutant. High levels of expression of sodBI and sodBII fully complemented the MD-hypersensitive phenotype of the triple sod null mutant, while sodBIII could only partially protect the mutant from MD toxicity (Fig. 6A). We extended the investigation by removing the signal sequence of pSodBIII, giving pSodBIII{Delta}29 (S. Mongkolsuk, P. Saenkham, and P. Vattanaviboon, unpublished data), which produces SodBIII in the cytoplasm. As shown in Fig. 6A, the expression of cytoplasmic SodBIII from pSodBIII{Delta}29 restored the MD resistance of the triple sod null mutant back to the wild-type level. Redox-cycling agents such as MD generate superoxide anions predominantly in the cytoplasm but also in the periplasm (53). In the cytoplasm, superoxide anion toxicity primarily arises from oxidation of iron sulfur clusters in enzymes (such as aconitase [20]) and the transcription factor FNR (45). This is consistent with our observations that cytoplasmic Sods fully protected the bacteria from MD toxicity. Nonetheless, the oxidation of periplasmic proteins provides a minor contribution to MD resistance levels, as shown by the ability of sodBIII to provide low-level MD resistance to the triple sod null mutant.

The role of sod genes in tumorigenesis. sods are virulence factors in some pathogenic bacteria (32, 40). Increased production and accumulation of ROS by host plants in response to bacterial invasion is an important part of the active plant defense response. Moreover, plants produce compounds which could cause an increased production of intracellular ROS (50). However, the role of ROS in the plant defense response against A. tumefaciens invasion is not clear. Recently, a catalase-peroxidase mutant has been shown to be attenuated in its ability to cause crown gall (52). This observation indicates that ROS play important roles in the A. tumefaciens/plant interaction and tumorigenesis. Thus, we tested various sod mutants containing the pTiC58 plasmid pCMA1 (27) for their abilities to induce tumors on tobacco leaf discs (44). The sodBI mutant was markedly reduced (80%) in its ability to induce tumors on the plant leaf discs (Fig. 6B and Table 3). Inactivation of either sodBII or sodBIII did not affect the ability to induce tumor formation (Fig. 6B and Table 3). However, the triple sod null mutant was avirulent, producing no tumors on leaf discs. Expression of sodBI, sodBII, and the cytoplasmic form of sodBIII (pSodBIII{Delta}29) restored the tumorigenicity to the triple sod null mutant (Fig. 6B and C). Unexpectedly, the periplasmic sodBIII (pSodBIII) failed to complement the phenotype of the mutant. This observation suggests that cytoplasmic Sods have important roles in the virulence of A. tumefaciens (Fig. 6C and Table 3). The abilities of various sod mutants to form tumors and of different sod genes to complement the avirulent phenotype show a pattern similar to the MD resistance phenotype. A reduced induction of vir genes has been shown to cause the attenuated tumor-forming phenotype of A. tumefaciens mutant strains (44). Thus, we tested whether a triple sod null mutant was defective in the induction of vir genes. pSM243cd and pSM358cd plasmids, which contain virB and virE promoter-lacZ fusions, respectively (44), were introduced into the sod null mutant harboring pCMA1 and its wild-type parent, NTL4/pCMA1, and the ability of acetosyringone to induce vir promoters was determined. As shown in Fig. 7A and B, the uninduced promoter activities of virB and virE were similar in both NTL4 and the triple sod null mutant (sodBI sodBII sodBIII triple mutant). Treatment with 100 µM acetosyringone induced the virB promoter by 23-fold in NTL4, while induction was 14-fold in the triple sod null mutant (Fig. 7A).


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  		TABLE 3. Virulence assay of A. tumefaciens strains on tobacco leaf explants


Figure 7
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  		FIG. 7. Expression of virB and virE in A. tumefaciens strains. A. tumefaciens NTL4, the sodBI sodBII sodBIII mutant (null), and the null mutant harboring pSodBI plasmid (null/pSodBI) containing a virB::lacZ fusion (A) or a virE::lacZ fusion (B) were incubated in induction broth (44) with (gray bars) or without (white bars) acetosyringone for 24 h. β-Galactosidase activities were measured.

A similar twofold decrease in acetosyringone induction of the virE promoter was detected for the mutant compared to what was seen for the wild-type parent (Fig. 7B). pSodBI could partially complement the decrease in acetosyringone-mediated induction of the virB and virE reporter fusions (Fig. 7A and B). The reduction in acetosyringone induction of vir promoters is at least in part responsible for the attenuated virulence of the Sod-deficient mutant (44).

Clearly, cytoplasmic Sod activity is needed for the full virulence of A. tumefaciens (Fig. 6C and Table 3). The role of Sod in tumorigenesis is consistent with a previous observation that virulent strains of A. tumefaciens have Sod activity higher than that of avirulent strains (4). A strain that lacks Sod likely accumulates intracellular superoxide anions. Many enzymes containing iron-sulfur clusters are extremely sensitive to superoxide anion inactivation due to oxidation and leaching of iron from the cluster. The inactivation of key members of the tricarboxylic acid cycle enzymes such as aconitase and fumarase in the triple sod null mutant probably contributes to the avirulent phenotype. A recent study has shown that the inactivation of citrate synthases in A. tumefaciens attenuates tumorigenesis (44). The lack of bacterial virulence could arise from defects in the mutants' abilities to synthesize branched-chain, sulfur-containing, and aromatic amino acids as a consequence of losing sod function (8). Alternatively, Sod may be an important factor for A. tumefaciens survival from the oxidative burst generated during the infection of plant wound sites. Studies with Xanthomonas campestris reveal the induction of sod expression during bacterial invasion of its host (42), suggesting the possible role of Sod in the infection.

High levels of superoxide anions are highly toxic to bacteria. A. tumefaciens has evolved and maintained three sods, each with different expression patterns and two cellular locations. The three superoxide dismutation systems consist of, first, the sodBI system, which is a constitutively highly expressed gene that functions as a housekeeping gene. sodBI is the major protector of the bacteria against superoxide stress. The second system involves the superoxide-inducible expression of sodBII. The major function of this enzyme is most likely during conditions under which intracellular superoxide anions briefly accumulate, thus leading to the oxidation of SoxR and the subsequent activation of sodBII to remove the excess superoxide anions. In the third system, sodBIII is expressed only during stationary phase, and the enzyme appears to localize to the periplasm. Its major role is probably to protect proteins in the electron transport chains and other proteins in the periplasmic space from damage by superoxide anions during the stationary phase of growth. These systems are complementary to each other and not redundant. Overall, they ensure that superoxide anions are kept at physiologically safe levels throughout exponential growth and stationary phase and during interaction with the host plant.


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ACKNOWLEDGMENTS
 
This research was supported by a research team strengthening grant from the National Center for Genetic Engineering and Biotechnology to S.M. and by a grant from the ESTM under the Higher Education Development Project of the Ministry of Education. P.S. was supported by Royal Golden Jubilee Scholarship PHD/0086/2546 from the Thailand Research Fund.

We thank S. Winans for pVIK112 and Peerakarn Banjerdkij for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Phone: (662) 574 0623, ext. 3816. Fax: (662) 574 2027. E-mail: skorn{at}cri.or.th Back

{triangledown} Published ahead of print on 5 October 2007. Back

{dagger} Present address: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101. Back


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Journal of Bacteriology, December 2007, p. 8807-8817, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.00960-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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