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Journal of Bacteriology, September 2007, p. 6477-6481, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00623-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Physiological and Expression Analyses of Agrobacterium tumefaciens trxA, Encoding Thioredoxin{triangledown}

Paiboon Vattanaviboon,1* Weerachai Tanboon,1 and Skorn Mongkolsuk1,2,3

Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210,1 Department of Biotechnology,2 Center for Emerging Bacterial Infections, Faculty of Science, Mahidol University, Bangkok 10400, Thailand3

Received 23 April 2007/ Accepted 6 June 2007


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ABSTRACT
 
Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H2O2 resistance phenotype.


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TEXT
 
Agrobacterium tumefaciens is a soilborne gram-negative pathogenic bacterium causing crown gall tumors in many dicotyledonous plants (29). As a plant pathogen and soil bacterium, A. tumefaciens is exposed to oxidative stress generated from aerobic metabolism, chemicals in the environment, and plant-generated oxidative bursts during plant-microbe interactions (16). Oxidative stress is highly toxic to bacterial cells, and they have evolved both enzymatic and nonenzymatic pathways to directly detoxify reactive oxygen species and repair oxidative stress-induced damage. This response involves the well-orchestrated coordination of gene expression governed by multiple transcription regulators.

Thioredoxins are small, ubiquitous, and evolutionarily conserved proteins involved in numerous biochemical processes (11). Thioredoxin has the ability to reduce disulfide bonds in target proteins. The two redox-active cysteine residues can be oxidized to form a disulfide and rereduced by the flavoenzyme thioredoxin reductase in an NADPH-dependent reaction. In its function as a thiol:disulfide reductant, thioredoxin, together with glutathione, contributes to the defense against oxidative stress by reducing disulfide bonds in oxidized proteins (4, 25). Thioredoxin is also an electron donor for several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase, and thiol peroxidases (3, 24). Moreover, the thioredoxin system plays important regulatory roles in cells (17, 33).

Several stresses have been shown to induce thioredoxin expression in prokaryotes. In Escherichia coli and Rhodobacter capsulatus, trxC, encoding thioredoxin 2, is a member of the peroxide-inducible OxyR regulon (26, 34). Moreover, induction of thioredoxin synthesis by oxidative stress has been observed in other bacteria (14, 18, 27, 30).

In this communication, we report the expression analysis and physiological characterization of trxA in A. tumefaciens. trxA has important roles in oxidative stress protection.

A. tumefaciens trxA. An annotated open reading frame, Atu0022, on the A. tumefaciens C58 circular chromosome encodes a 106-amino-acid protein product of 11.1 kDa identified as TrxAC1 (32). Its deduced primary structure has 49 and 50% identity to Bacillus subtilis (5) and E. coli (12) TrxA, respectively (data not shown). The highly conserved thioredoxin active site, Trp-Cys-Gly-Pro-Cys-Lys, is conserved in Atu0022. Atu0022 likely encodes a TrxA protein and is henceforth designated trxA. A. tumefaciens trxA is located next to folC (Atu0021), a convergently transcribed gene encoding a homolog of dihydrofolate synthase. The gene upstream of trxA was annotated as uvrD (Atu0023), encoding DNA helicase, an important enzyme in DNA repair and replication. uvrD and trxA are transcribed in the same orientation and are separated by a 77-bp region containing the promoter of trxA. The folC-trxA-uvrD gene organization is conserved in several Alphaproteobacteria (data not shown). Many bacteria have two trx genes; thus, an E. coli thioredoxin 2 amino acid sequence encoded by trxC (20) was used to search with BlastP (2) the A. tumefaciens C58 genome (10, 32). No close homologs of the protein could be identified.

Oxidative stress-induced trxA expression. Bacterial trxA has complex expression patterns. Heat, osmotic, and oxidative stresses have been shown to induce trxA expression (14, 18, 27, 30). Hence, trxA expression in response to oxidants and a thiol-depleting agent was determined by Northern analysis. RNA samples extracted from A. tumefaciens NTL4 (19) exponential-phase cultures uninduced and induced with 250 µM H2O2, 200 µM diamide (DA) (a thiol-oxidizing agent), 200 µM cumene hydroperoxide (CHP), and 200 µM menadione (MD) were used. The Northern analysis results reveal a positive hybridization band of 360 bases with the trxA probe. This corresponds to monocistronic trxA transcripts (Fig. 1A). The levels of trxA mRNA show large increases in response to MD treatment (12-fold, as judged from densitometric analysis) and to a lesser extent in response to treatment with DA (7-fold) and CHP (5-fold). By contrast, treatment with 250 µM H2O2 did not induce trxA expression. The results indicate that trxA expression is under oxidative stress regulation and reflect the need of A. tumefaciens for thioredoxin molecules in the cells during oxidative stress. The inability of H2O2 to induce trxA expression suggests that, unlike in other bacteria, A. tumefaciens TrxA might not be required as a cofactor for H2O2 metabolism and/or protection. The stress-inducible regulation of trxA expression is an important strategy enabling A. tumefaciens to adjust the intracellular level of thioredoxin so that proper cellular functions under stressful conditions can be maintained.


Figure 1
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  		FIG. 1. Expression analysis and promoter localization of A. tumefaciens trxA. (A) Northern blot of total RNA (10 µg) samples prepared as previously described from A. tumefaciens exponential-phase cultures (7): uninduced (UN) and induced with 250 µM H2O2, 200 µM CHP, 200 µM MD, and 200 µM DA for 15 min. The RNA samples were separated, blotted, and hybridized with a 32P-labeled trxA-specific probe. 16S rRNA levels were used as loading controls and are shown below the autoradiograph of the Northern blot. The smaller RNA bands running below the 16S rRNA were the result of processing of the 23S rRNA. (B) Primer extension of RNA extracted from uninduced (UN) and CHP- and MD-induced cultures. Primer extension experiments were performed as previously described (7) with the 32P-labeled oligonucleotide primer BT1562 (5' CCCAGAAATCCACCACGAC 3'). The DNA sequence ladders G, A, T, and C were generated by a PCR sequencing kit with labeled pUC/M13 forward primer and pGEM-3Zf (+) as the template (Applied Biosystems). Numbers next to the ladder represent the length of the fragment from the primer. The arrowheads labeled P1 and P2 indicate trxA transcription start sites. The –10 and –35 promoter regions of P1 (black) and P2 (gray) are shown in bold and underlined. A putative ribosome binding site (Rbs) is shown in italics.

In various bacteria, diverse transcriptional regulators are involved in regulation of trx expression. The observed patterns of oxidants inducing A. tumefaciens trxA expression have similarity to patterns of A. tumefaciens OxyR (a H2O2 sensor/transcription regulator)-regulated genes (21). In addition, SoxR (7) and OhrR (6) are members of oxidative stress-responsive transcription regulators that sense increased levels of superoxide anions and organic hydroperoxides, respectively. Northern analyses of MD and CHP induction of trxA expression in A. tumefaciens NTL4 oxyR, soxR, and ohrR mutants were performed. The results in Fig. 1A illustrate that induction of trxA expression by MD and CHP is independent of the known oxidative sensor transcription regulators OxyR, SoxR, and OhrR. Oxidant-induced expression of trxA is likely to be regulated by a novel unknown oxidant sensor/transcription regulator. This possibility is being investigated.

Characterization of the trxA promoter. Primer extension analysis was performed to localize the 5' end of trxA mRNA and the transcription start site of trxA with total RNA from bacteria grown under uninduced and CHP- and MD-induced conditions. Two primer extension products were identified (Fig. 1B). The majority of trxA transcripts initiated at a G residue situated 29 nucleotides upstream of the assumed trxA ATG codon. E. coli RNA polymerase {delta}70 consensus –10- and –35-like motifs were identified as TCACAT and TTGAAA, respectively, and were separated by 16 bp (Fig. 1B). The trxA promoter architecture resembles typical strong promoters in A. tumefaciens (6, 7). An additional minor 5' end of the trxA mRNA was mapped to a T residue located 10 nucleotides downstream of the major trxA transcription start site. The putative –35 (TTCGAC) and –10 (GATAGC) regions were atypical of A. tumefaciens promoters, a finding which is reflected in the low levels of transcription initiation for this minor promoter. Alternatively, the minor 5' end of trxA mRNA could arise from premature termination of reverse transcriptase. The primer extension results indicated that both the trxA major promoter and the putative trxA minor promoter were induced by CHP and MD pretreatments. The putative binding sites for the A. tumefaciens oxidative stress sensor transcription regulators OxyR, OhrR, and SoxR have been reported (6, 7, 21). The binding sites for these regulators could not be found in the vicinity of either the major or minor trxA promoter; this result further supports the Northern blot analysis of trxA expression, which shows the gene to be regulated by a novel unknown regulator.

Physiological analysis of the trxA mutant. In many bacteria, inactivation of trxA has resulted in pleiotropic changes in bacterial phenotypes, including hypersensitivity to stresses, growth defects, and cell death (4, 14, 25, 27). In order to evaluate the function of trxA in A. tumefaciens, a trxA mutant was constructed by targeted insertion inactivation of the gene by pKnocktrxA, performed as previously described (1, 6), and the mutant was confirmed by Southern analysis. First, we examined the growth rate of the trxA mutant and the isogenic wild-type strain NTL4 grown in a rich medium (Luria-Bertani [LB]). The trxA mutant displayed a growth defect in LB medium by a prolonged exponential phase, with the mutant having a doubling time of 99 min, compared with 83 min for the NTL4 parental strain (data not shown). Moreover, the trxA mutant showed a defect in the aerobic plating efficiency. The mutant had an aerobic plating efficiency 2 orders of magnitude lower than that of NTL4 (Fig. 2A). However, when the plating efficiency was assessed under a microaerobic atmosphere (6% oxygen and 12% carbon dioxide) generated from the CampyGen system (Oxoid, United Kingdom), the mutant plating efficiency was less than 1 order of magnitude lower than that of the wild type (data not shown). This suggests that oxygen under aerobic conditions caused the reduced plating efficiency of the trxA mutant. Both the retarded growth rate and reduced plating efficiency phenotypes of the trxA mutant could be completely restored by trans expression of trxA from pTrxA (trxA in the broad-host-range plasmid vector pBBR1MSC-2 [15]), as illustrated in Fig. 2A (trxA/pTrxA), indicating that these phenotypes resulted from lack of thioredoxin. Thus, we further tested the ability of dithiothreitol (DTT), a disulfide reductant capable of crossing membranes, to complement the growth defect phenotypes of the trxA mutant. The results show that addition of 100 µM to 5 mM DTT to the culture medium failed to alleviate growth defects in the trxA mutant (data not shown), suggesting that the inability to reduce disulfide bonds of cytoplasmic proteins and small molecules such as oxidized coenzyme A and oxidized glutathione were not key factors in causing the growth defect in the trxA mutant.


Figure 2
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  		FIG. 2. Plating efficiency and oxidase tests of A. tumefaciens strains. (A) Exponential-phase A. tumefaciens cultures in LB medium were adjusted to an optical density at 600 nm of 0.1 before 10-fold serial dilutions were made. Then, 10 µl of each dilution was spotted onto an LB agar plate and incubated at 30°C for 24 h. (B) Quantitative analysis of cytochrome c oxidase activity was measured as previously described (28). Essentially, 10 µl of clear lysate was added to 940 µl of assay buffer (10 mM Tris-HCl [pH 7.0], 120 mM KCl). The reaction was initiated by the addition of 50 µl of reduced cytochrome c solution (218 µM in 0.5 mM DTT), and the reduction in the A550 was measured kinetically for 1 min. A unit of cytochrome oxidase activity is expressed as the amount required to oxidize 1 µmol of ferrocytochrome c per min at 25°C and pH 7.0. The calculation is based on the fact that the difference in the extinction coefficient ({Delta}{varepsilon}mM) between ferrocytochrome c and ferricytochrome c at 550 nm is 21.84 (28).

Thioredoxin is involved in many enzymatic systems and has diverse functions, both in cellular processes and antioxidant defenses (4). The reduced aerobic plating efficiency phenotype has been shown previously to be associated with defects in antioxidant defense systems that cause accumulation of oxidants and subsequent cellular damage. This led us to test whether high-level expression of genes encoding antioxidant enzymes from an expression vector could rescue the aerobic growth defects of the trxA mutant. High levels of the Agrobacterium catalase-peroxidase KatA (trxA/pKatA) (22), organic hydroperoxide resistance peroxidase (trxA/pOhr) (6), and a cytoplasmic iron superoxide dismutase (SOD) (trxA/pSod1) (S. Mongkolsuk et al., unpublished data) partially rescued the reduced plating efficiency phenotype of the trxA mutant (Fig. 2A). It appears likely that the reduced aerobic plating efficiency phenotype was at least in part due to intracellular accumulation of different types of reactive oxygen species, including H2O2, organic hydroperoxides, and superoxide anions. In addition, decreased levels of some essential reductase enzymes that require thioredoxin for their activities, such as ribonucleotide reductase, could have adverse effects on bacterial growth (25). Methionine sulfoxide reductase, an enzyme that repairs oxidized proteins, also requires thioredoxin as a reducing equivalent. Thus, reduction in these enzyme activities could be partially responsible for the trxA phenotype.

TrxA not only is involved in the reduction of protein disulfides in the cytoplasm but also is a component of the electron transfer pathway from the cytoplasm to the periplasmic thiol-disulfide oxidoreductases, a cascade of enzymes responsible for disulfide bonding of periplasmic proteins and maturation of cytochrome c (8). Inactivation of trxA renders E. coli unable to assemble c-type cytochrome (23). Hence, we determined whether TrxA was required for cytochrome c assembly by measuring the cytochrome c oxidase activity (28) in the A. tumefaciens strains. As shown in Fig. 2B, the trxA mutant produced drastically reduced cytochrome c oxidase activity (0.3 ± 0.2 U mg of protein–1 [mean ± standard deviation]) compared with the isogenic wild-type strain NTL4 (16.8 ± 0.9 U mg of protein–1) and the mutant complemented with pTrxA (15.0 ± 2.4 U mg of protein–1). Although pKat, pSod1, and pOhr partially suppressed the growth defects of the mutant, they failed to complement the cytochrome c oxidase deficiency of the mutant (Fig. 2B), indicating that the reduced aerobic plating efficiency and oxidase deficiency could arise from different pathways.

Because we identified A. tumefaciens trxA by its homology to known thioredoxin genes, an argument could be raised concerning whether the observed phenotypes in the A. tumefaciens trxA mutants are actually due to the mutants’ inability to produce thioredoxin. We use the rationale that if various phenotypes of the A. tumefaciens trxA mutant arose from lack of thioredoxin, they should be complemented by a well-characterized thioredoxin gene from other bacteria. Thus, an expression plasmid, pEcotrxA, which contains the well-characterized E. coli trxA gene (12) in the broad-host-range vector pBBR1MSC-2 (15), was transformed into the trxA mutant, and the ability to complement various phenotypes of the mutant was tested. The results in Fig. 2A and B show that expression of E. coli trxA restored the altered phenotypes of the trxA mutant. This confirms that the observed trxA mutant phenotypes result from inactivation of trxA.

Alteration of oxidative stress resistance in the trxA mutant. Thioredoxin is important to many antioxidant enzymes. Thus, the resistance levels against various oxidants in the A. tumefaciens trxA mutant and its parental strain, NTL4, were determined by the inhibition zone method. As the mutant showed growth defects under an aerobic atmosphere, the oxidant resistance tests were performed under microaerobic conditions (6% O2 generated from CampyGen [Oxoid, United Kingdom]) to minimize growth rate differences between the strains. We found that under this condition, the defect in bacterial growth of the trxA mutant has no significant effects on the tests, as shown by all strains having similar zones of inhibition against imipenem antibiotic disks (30 µg) (Oxoid, United Kingdom). For the parental strain, the zones of growth inhibition for H2O2 (1.0 M), MD (1.0 M), CHP (1.0 M), and DA (1.0 M) were 13.2 ± 0.6, 18.2 ± 0.5, 22.0 ± 0.4, and 28.2 ± 0.8 mm, respectively, compared to zones of 10.0 ± 0.4, 21.3 ± 0.7, 22.5 ± 0.6, and 28.0 ± 0.7 mm, respectively, for the trxA mutant (Fig. 3A). The phenotypes of increased resistance to H2O2 and decreased resistance to MD in the trxA mutant could be complemented by expression of trxA from pTrxA (Fig. 3A).


Figure 3
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  		FIG. 3. Determinations of oxidant resistance in A. tumefaciens strains. (A) The resistance levels to oxidants of A. tumefaciens NTL4, the trxA mutant, and the mutant complemented with trxA (trxA/pTrxA), sod1 (trxA/pSod1), and sod2 (trxA/pSod2) on the expression vectors were measured by zones of growth inhibition around 6-mm-diameter paper disks (prepared from Whatman filter paper no. 3) soaked with 5 µl of 1 M H2O2, 1 M CHP, 1 M MD, and 1 M DA, as described previously (21). Inhibition zones were measured after 24 h of incubation at 30°C under a microaerobic atmosphere generated from CampyGen (Oxoid, United Kingdom). Values are means and standard deviations of four replicates. (B) Resistance levels to oxidants of the A. tumefaciens oxyR mutant and the oxyR trxA double mutant against 0.5 M H2O2 and 0.5 M MD.

Menadione is thought to exert its toxicity by generating intracellular superoxide anions via a redox cycling reaction (9). Hence, the levels of total SOD activity in the trxA mutant were determined (21). The total SOD level in the trxA mutant was 0.28 ± 0.05 U mg of protein–1, similar to that found in the isogenic wild-type strain NTL4 (0.27 ± 0.06 U mg of protein–1). Thus, the decreased MD resistance phenotype of the trxA mutant is independent of SOD activity. In addition, inactivation of trxA resulted in defects in cytochrome c maturation (Fig. 2B). The disruption of electron transfer could be responsible for the leakage of electrons from the respiratory chain, leading to increased production of superoxide anions (13). Consequently, the trxA mutant generates and accumulates more intracellular superoxide anions, which renders it hypersensitive to a superoxide generator, MD. We attempted to verify this assumption by introducing sod1, a major cytoplasmic iron SOD (FeSOD) (pSod1), and sod2, a periplasmic FeSOD (pSod2), on the expression vector pBBR1MSC-2 into the trxA mutant, and we determined the MD resistance levels in the transformants. The results show that neither pSod1 nor pSod2 was able to complement the decreased MD resistance phenotype of the mutant (Fig. 3B). Thus, accumulation of intracellular superoxide anions is not involved in the phenotype of increased sensitivity to superoxide, even though it is partially responsible for aerobic growth defects.

Increased resistance to H2O2 in the trxA mutant was unexpected. Generally, the level of H2O2 resistance is directly correlated with total catalase activity, as is also the case in A. tumefaciens (31). The trxA mutant had total catalase activity of 7.7 ± 0.6 U mg of protein–1, which was comparable to the enzyme level in the parental strain NTL4 (7.5 ± 0.7 U mg of protein–1). OxyR is directly involved in determining the H2O2 resistance in A. tumefaciens (21); hence, we tested whether it had a role in the increased H2O2 resistance in the trxA mutant. A trxA-oxyR double mutant was constructed by introducing pKNOCKoxyR (21) into the trxA mutant. The double mutant was more resistant to H2O2 than was the oxyR single mutant (Fig. 3B). Thus, the increased H2O2 resistance phenotype of trxA is independent of OxyR-regulated genes and the catalase enzyme. However, the finding that overexpression of catalase partially compensated for the reduced plating efficiency of the trxA mutant suggested that the mutant does accumulate intracellular H2O2 (Fig. 2A). Inactivation of trxA could lead to increased expression of OxyR-independent genes, which are involved in repaired or limited H2O2-induced damages to macromolecules. It is likely that the high resistance to external H2O2 of the trxA mutant is not due to enhanced H2O2 degradation.

Conclusions. A. tumefaciens trxA, encoding thioredoxin, has important roles in a variety of cellular functions. The expression of trxA is oxidative stress inducible and regulated by unknown regulators of the oxidative stress response. Inactivation of trxA severely affected bacterial growth under the conditions of an aerobic atmosphere and cytochrome c maturation. The mutant had unusual oxidative stress sensitivity phenotypes, being more sensitive to MD and less sensitive to H2O2 than was the wild type.


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ACKNOWLEDGMENTS
 
This research was supported by a Research Team Strengthening Grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC) to S.M. and by grants from the ESTM through the Higher Education Development Project of the Commission on Higher Education, Ministry of Education.

We thank Supa Utamapongchai for technical assistance and James M. Dubbs for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Phone: 662 574 0622, ext. 3815. Fax: 662 574 2027. E-mail: paiboon{at}cri.or.th Back

{triangledown} Published ahead of print on 15 June 2007. Back


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Journal of Bacteriology, September 2007, p. 6477-6481, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00623-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Saenkham, P., Eiamphungporn, W., Farrand, S. K., Vattanaviboon, P., Mongkolsuk, S. (2007). Multiple Superoxide Dismutases in Agrobacterium tumefaciens: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis. J. Bacteriol. 189: 8807-8817 [Abstract] [Full Text]  

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