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Journal of Bacteriology, March 2007, p. 2110-2113, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01802-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Novel Sinorhizobium meliloti Mutants Compromised for Oxidative Stress Protection and Symbiosis{triangledown}

Bryan W. Davies and Graham C. Walker*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 29 November 2006/ Accepted 8 December 2006


   ABSTRACT
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Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both sensitive to hydrogen peroxide and symbiotically defective on the host plant Medicago sativa. The mutations affect a wide variety of cellular processes and represent both novel and previously identified genes important in symbiosis.


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During symbiotic development with the host plant Medicago sativa, Sinorhizobium meliloti is subjected to a prolonged oxidative burst released by its host (33). The burst is composed of at least superoxide and hydrogen peroxide (H2O2), both of which are deleterious to cell survival. The prolonged burst is a chronic stress for S. meliloti, with both superoxide and H2O2 readily detected in nodules several weeks after infection (33). It has become apparent that S. meliloti must be able to manage oxidative stress while in its host, but the issue is complex. For example, S. meliloti single mutants that lack either catalase A (katA) or catalase C (katC), both of which detoxify H2O2, are symbiotically proficient, whereas the katA katC double mutant is symbiotically defective. Furthermore, novel regulatory mechanisms may control the S. meliloti response to oxidative stress in planta as the disruption of the major regulator of H2O2 defense in the free-living bacterium, oxyR, does not adversely affect symbiosis (18). Given the obvious necessity of managing oxidative stress in the plant and the apparent complexity of the defense network required to meet this stress, we hypothesized that there might be additional oxidative stress defense mechanisms outside the spectrum of classic defense enzymes required for symbiosis. To explore this hypothesis, we undertook a novel two-part screen to identify S. meliloti mutants that are both sensitive to oxidative stress in the free-living state and symbiotically defective. Through this screen we identified several genes that have not previously been recognized as being important either in symbiosis or in oxidative stress protection for S. meliloti.

Identification of H2O2-sensitive and symbiotically defective S. meliloti mutants. S. meliloti is exposed to chronic H2O2 stress in the infection thread and nodule (33). Previous work has shown that certain S. meliloti strains compromised for H2O2 detoxification are symbiotically defective (19, 34). Considering this work, we chose to use H2O2 as the oxidative stress agent with which to screen S. meliloti mutants. We obtained a random pool of S. meliloti mutants by mutagenizing the wild-type strain, Rm1021, with mTn5-GusNm (mTn5) (2, 30). mTn5 was introduced into Rm1021 on a plasmid by triparental mating (10, 30). mTn5 mutants were selected on Luria-Bertani (LB) agar plates containing 200 µg/ml neomycin at 30°C. We measured the endogenous peroxide level of the LB agar by using the Amplex Red hydrogen peroxide/peroxidase assay kit (Molecular Probes) and found it to be 1.8 µM. Since a sublethal dose of H2O2 for wild-type S. meliloti is 1 mM, we felt confident that the endogenous peroxide levels of the LB agar would not perturb our study (18). Mating cultures were diluted so as to obtain approximately 100 colonies per plate, and H2O2-sensitive mutants were subsequently identified by replica plating onto LB plates containing 0.7 mM H2O2 and 10 mM HEPES, pH 7.0. After 3 days, we identified colonies unable to grow on plates containing H2O2. H2O2 sensitivity was confirmed by zone-of-inhibition assays (7). The mTn5 mutants showing sensitivity to H2O2 were tested for symbiotic proficiency with the plant host M. sativa. After 4 weeks, plant height was measured and compared to that of Rm1021-inoculated plants. Mutant-inoculated plants that were statistically shorter than Rm1021-inoculated plants were initially considered to be symbiotically defective. To confirm that mTn5 was linked to both the H2O2 sensitivity and symbiotic deficiency, each mutant allele was transduced back into the parental strain Rm1021 (12). Three independent transductants for each mutant allele were tested for both H2O2 sensitivity and symbiotic proficiency. To more precisely analyze the symbiotic phenotype, nitrogenase activity was then determined using acetylene reduction (37).

Santos et al. showed that S. meliloti is exposed to chronic oxidative stress while in the plant (33). Furthermore, Herouart et al. (15). demonstrated that S. meliloti mutants defective in katA are sensitive to acute oxidative stress but do not show a symbiotic defect. These results suggest that chronic resistance to oxidative stress may be an important factor for S. meliloti to establish functional symbiosis. Because of these observations, we employed assays such as zone of inhibition that measure S. meliloti's sensitivity to chronic oxidative stress. In total, we screened 1.5 x 104 mTn5 mutants. After transduction, we isolated 112 H2O2-sensitive mutants. Of these, nine mutants were also symbiotically defective. To more broadly characterize the oxidative stress sensitivity of these mutants, we tested them for chronic sensitivity to superoxide with a zone-of-inhibition assay using the superoxide generator menadione.

We used random primer PCR to identify the insertion point of mTn5 in each mutant (Table 1) (30). mTn5 has not been tested to determine if it contains the outward-reading promoter present in the parental Tn5 (4, 30). Consequently, the insertion of the mTn5 may cause polar effects on downstream genes; however, previous work from our lab shows that the expression of downstream genes can occur in at least some contexts with mTn5 (8).


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  		TABLE 1. Rm1021 mTn5 mutants identified as being both H2O2 sensitive and symbiotically defective

 
The exoP::mTn5, glgA1::mTn5, and sitA::mTn5 mutants all show small increases in sensitivity to H2O2 in zone-of-inhibition assays (Table 1). To better quantify the H2O2 sensitivities of these mutants, cultures of each mutant strain and Rm1021 were serially diluted and spread onto LB plates and LB plates containing 0.3 mM H2O2 to determine the relative plating efficiencies of the strains. After 3 days of growth, visible colonies were counted. Using this chronic stress method, we determined that the exoP::mTn5, glgA1::mTn5, and sitA::mTn5 mutants show plating efficiencies of 2.1% ± 0.2%, 11.3% ± 2.3%, and 4.6% ± 2.1%, respectively, relative to Rm1021 on LB plates containing 0.3 mM H2O2.

Defects in genes associated with succinoglycan production cause H2O2 sensitivity. Rm1021 produces an acidic exopolysaccharide, succinoglycan, that is required for proper symbiotic development (23). Succinoglycan is produced in both high- and low-molecular-weight forms and carries succinyl, acetyl, and pyruvyl modifications (1, 31). The low-molecular-weight fraction of succinoglycan is of particular interest because past studies have reported that the low-molecular-weight succinoglycan, rather than the high-molecular-weight succinoglycan, is able to restore the ability of invasion-deficient mutants to invade nodules (3, 38, 40). Our screen identified three genes previously shown to be required for succinoglycan production and symbiosis (Table 1) (14).

ExoP is required for polymerization of the succinoglycan monomer. It is thought to work in conjunction with ExoQ to produce high-molecular-weight succinoglycan or with ExoT to produce the low-molecular-weight form (14, 32). A role for succinoglycan in protection against oxidative stress in S. meliloti has not previously been reported. One possibility is that succinoglycan can act as a diffusion barrier against H2O2, thereby protecting cells against exogenous H2O2. Consistent with this hypothesis, we found that an exoY mutant, which completely lacks succinoglycan, shows increased sensitivity towards H2O2 (zone of inhibition = 5.9 ± 0.1 cm) (32). This hypothesis is supported by results from studies of the nodulation of Sesbania rostrata by Azorhizobium caulinodans. The early stages of this symbiosis are characterized by a massive production of H2O2 by the plant host. In situ H2O2 localization demonstrated that increased exopolysaccharide production by A. caulinodans prevents the incorporation of H2O2 inside the bacterium, suggesting a role for exopolysaccharide in protecting A. caulinodans against H2O2 (11). Additionally, the extensive pyruvyl modifications on Rm1021 succinoglycan may play a role in protection against H2O2 as pyruvate has been shown to scavenge H2O2 nonenzymatically (39). The sensitivity of these exopolysaccharide mutants appears to be specific to H2O2 as none of the exopolysaccharide mutants showed increased sensitivity to menadione relative to Rm1021 (Table 1).

We also identified exoD mutants in our screen. Although mutations in exoD lead to altered succinoglycan production, no biosynthetic role in succinoglycan synthesis has been attributed to exoD. Furthermore, genetic evidence has shown that altered production of succinoglycan is not the cause of the symbiotic defect in an exoD mutant (28). Although efforts have been made to define a physiological function for ExoD, further research will be required to explain why the loss of this gene causes increased H2O2 sensitivity.

Metabolic defects cause H2O2 sensitivity and symbiotic defects. Our screen identified mutations in several different metabolic pathways, including nucleoside biosynthesis and sugar storage and metabolism (Table 1). We identified a strain with a defect in tkt-2, which encodes one of two paralogs of transketolase found in the Rm1021 genome. This mutant is sensitive to both H2O2 and menadione. Transketolase functions at two stages in the nonoxidative steps of the pentose phosphate pathway (41). Enhanced flux of sugars through the pentose phosphate pathway has been linked to oxidative stress resistance, possibly by increasing the production of reducing power in the form of NADPH (5, 17, 20).

Another mutant which is both sensitive to H2O2 and symbiotically defective has an mTn5 insertion in glgA1, which encodes a putative glycogen synthase that adds glucose to growing starch chains. In S. meliloti, glgA1 lies directly upstream of the gene for phosphoglucomutase (pgm). pgm is also an exo gene (exoC) which catalyzes the reversible conversion of glucose-1-phosphate into glucose-6-phosphate for entry into carbon metabolism. S. meliloti exoC mutants induce empty, ineffective nodules on alfalfa and are dim on calcofluor due to a deficiency in succinoglycan synthesis (23). Calcofluor is a dye that fluoresces under UV light when bound to certain ß-linked polysaccharides, such as succinoglycan (23). In contrast to exoC mutants, our glgA1 mutant was bright on calcofluor (data not shown) and indistinguishable from Rm1021, indicating that the mTn5 insertion in our glgA1 mutant was not polar on exoC. A role for glucose storage in oxidative stress protection in S. meliloti has not previously been reported, and further investigation will be required to elucidate this role. Interestingly, a glycogen synthase mutant of Rhizobium tropici shows enhanced symbiotic performance on the plant host Phaseolus vulgaris. This contrasting result highlights how different symbiotic associations can have significantly different host-symbiont requirements (26).

Our screen also identified a strain with a defect in purL, which encodes phosphoribosylformylglycinamidine (FGAM) synthetase, the fourth enzyme in the pathway for purine biosynthesis (24). Purine auxotrophs of most rhizobial species, including S. meliloti, have previously been shown to be symbiotically defective; however, they have not been reported to be sensitive to oxidative stress (6, 22, 25). It was recently shown that a Sinorhizobium fredii purL mutant has an altered lipopolysaccharide (LPS) layer, though the reasons for this remain unclear (6). We considered the possibility that an altered LPS layer might allow easier diffusion of H2O2 into the cell, thus explaining increased sensitivity. However, we found that, unlike that in S. fredii, the disruption of purL in S. meliloti does not cause a change in the LPS layer observable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (data not shown).

Defects in metal transport, protein biosynthesis, and cytochrome c biogenesis cause H2O2 sensitivity and symbiotic defects. SitA is the periplasmic binding protein of a putative Mn-Fe ABC transporter. Our investigation of the sitA::mTn5 mutant is discussed in the accompanying report (8).

We were very surprised to identify a peptidyl-tRNA hydrolase (pth) mutant in our screen since Rm1021 has only one copy of pth in its genome (13). Pth scavenges peptidyl-tRNA molecules that arise normally during protein biosynthesis, is ubiquitous among bacteria, and, in most cases, is an essential enzyme (16). Our pth::mTn5 mutant also shows considerable sensitivity to menadione, suggesting that Pth may have a general role in oxidative stress protection. One possibility is that translation increases in response to oxidative stress that in turn increases the demand on tRNA recycling.

The final gene we identified as being important in either symbiosis or oxidative stress protection was cycK. cycK is part of the cycHJKL operon involved in cytochrome c-type biosynthesis. cyc mutants of S. meliloti and other rhizobial species have been identified previously, and cyc has been reported to be required for symbiosis but not for oxidative stress protection (9, 21, 35). The cycK::mTn5 mutant exhibits the greatest increase in sensitivity to H2O2 and menadione (Table 1). In S. meliloti, c-type cytochromes are required for nitrate reduction ex planta and nitrogen fixation in root nodules (21). The role of cycK and cytochrome c in oxidative stress defense will require further investigation.

Among the 112 mTn5 mutants we identified that were H2O2 sensitive but symbiotically proficient, we identified the mTn5 insertion point for the three mutants that displayed the most severe H2O2 sensitivity (Table 2). The mutant exhibiting the greatest sensitivity had a disruption in the global regulator of H2O2 protection, oxyR, which has previously been shown not to be required for symbiosis (18). actR was originally identified in S. meliloti as part of a two-component system required for growth at low pH (36). Recent work with Escherichia coli has shown that pH changes and oxidative stress affect the regulation of a large and overlapping set of genes, suggesting a strong relationship between acid stress and oxidative stress (27). Interestingly, the actR::mTn5 mutant is also very sensitive to menadione, suggesting a general role in oxidative stress protection. We also identified a putative open reading frame (SMc01853) coding for a protein with a DnaJ domain. As oxidative stress causes protein damage, the SMc01853 protein may act as a chaperone to manage oxidized proteins.


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  		TABLE 2. Rm1021 mTn5 mutants that were symbiotically proficient but displayed high sensitivity to H2O2

 
Comparison of Tables 1 and 2 shows that the sensitivity of a mutant strain to H2O2 or menadione ex planta does not correlate with the strain's ability to effectively nodulate alfalfa. This finding suggests that, of the several oxidative stress defense systems available to S. meliloti, only a specific subset may be required to combat the oxidative stress encountered in planta. This may be due to the specific composition of the oxidative burst S. meliloti experiences in planta, which has not been fully characterized (33). Further study of Table 1 also shows a lack of correlation between the sensitivity of a strain to H2O2 or menadione ex planta and its capacity to fix nitrogen in planta. This may be because the oxidative stress sensitivity of some of the mutants is not the cause of their symbiotic deficiency. Alternatively, the different oxidative stress defense systems may be required at different developmental stages, allowing some mutants to proceed further in symbiosis than others. Our screen has identified several genes previously not associated with oxidative stress protection in S. meliloti. The results of this screen suggest that oxidative stress protection encompasses a much broader range of cellular functions than traditionally recognized.


   ACKNOWLEDGMENTS
 
We thank members of the Walker lab for careful review of the manuscript.

This work was supported by National Institutes of Health grant GM31010 to G.C.W. and a National Sciences and Engineering Research Council of Canada graduate scholarship to B.W.D. G.C.W. is an American Cancer Society Research Professor.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-6711. Fax: (617) 253-2643. E-mail: gwalker{at}mit.edu. Back

{triangledown} Published ahead of print on 15 December 2006. Back


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Journal of Bacteriology, March 2007, p. 2110-2113, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01802-06
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