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Journal of Bacteriology, March 2007, p. 2101-2109, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01377-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Disruption of sitA Compromises Sinorhizobium meliloti for Manganese Uptake Required for Protection against Oxidative Stress

Bryan W. Davies and Graham C. Walker^*

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

Received 29 August 2006/ Accepted 8 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Symbiosis with the gram-negative -proteobacterium Sinorhizobium meliloti allows the host plant, Medicago sativa (alfalfa), to utilize atmospheric nitrogen fixed by the microsymbiont. Through a complex exchange of chemical signals, S. meliloti induces root hair curling and nodule formation on alfalfa. S. meliloti organisms trapped in the curled root hairs invade M. sativa through a tube-like structure called an infection thread and are eventually released into the cells of the developing nodule, where they differentiate into nitrogen-fixing bacteroids (8, 16).

While in the infection thread, S. meliloti is exposed to an oxidative burst released by the host plant that is composed of at least hydrogen peroxide (H₂O₂) and superoxide (42). S. meliloti encodes a set of enzymes to defend against these reactive oxygen species (ROS), including superoxide dismutases, catalases, and alkylhydroperoxidases (17). It has become evident that S. meliloti must be able to manage oxidative stress while in the host plant, as the loss of certain oxidative stress defense mechanisms causes symbiotic defects. For example, S. meliloti strains deficient in both catalase B and catalase C (katB katC) or both catalase A and catalase C (katA katC), enzymes that detoxify H₂O₂, are symbiotically deficient (28, 44). However, exactly which rhizobial defenses are required to combat oxidative stress is a complex question. Several genes known to be required for defense against ROS in the free-living state are dispensable for symbiosis. For example, disruption of the global regulator of H₂O₂ protection, oxyR, makes the free-living strain extremely sensitive to H₂O₂ but does not affect symbiosis (27).

We were interested in identifying novel genes involved in S. meliloti oxidative stress protection and determining if these genes also play a role in the development of symbiosis. To explore this question, we undertook a two-part screen to identify transposon mutants of S. meliloti that were sensitive to H₂O₂ and that were also defective in symbiosis (12). One mutant identified in the screen has a transposon insertion in the sitA gene. sitA is the first gene in the four-gene operon sitABCD, which has been annotated as coding for a putative iron/manganese ABC transporter (17).

Iron and manganese are important metals for oxidative stress protection. Iron is used as a cofactor in defense enzymes such as catalase (4). Although in this context iron is helpful in protection against ROS, free iron in the Fe²⁺ state can serve to exacerbate oxidative stress by producing hydroxyl radicals from peroxides through Fenton chemistry (26). In contrast to iron, manganese ions can help defend against ROS by scavenging both H₂O₂ and superoxide, as part of low-molecular-weight complexes with cellular ligands such as phosphate, lactate, or bicarbonate. Although the exact chemistry of the scavenging has not been determined, the mechanism is thought to involve manganese ions cycling between the Mn²⁺ and Mn³⁺ states (2, 3, 24, 45). In its enzymatic capacity, manganese also aids in oxidative stress defense by acting as the essential cofactor in dedicated ROS-scavenging enzymes such as manganese-containing superoxide dismutases and catalases (5, 40).

In addition to safeguarding bacteria against ROS, manganese has also been shown to play an important role in virulence. Disruption of manganese uptake in pathogens Salmonella enterica serovar Typhimurium and Streptococcus pyogenes attenuates their virulence (7, 29). Furthermore, in S. pyogenes disruption of manganese uptake has also been linked to oxidative stress sensitivity (29). The use of manganese in virulence is most pointedly observed in the extreme case of the Lyme disease pathogen Borrelia burgdorferi. B. burgdorferi has dispensed with a requirement for iron and has evolved survival strategies that are fully accommodated by manganese (36).

Considering the known roles for manganese in oxidative stress protection and the requirement for manganese uptake in pathogen-host interactions, the sitA mutant we identified was an intriguing candidate for further exploration. To define the role of SitA in S. meliloti free-living and symbiotic physiology, we first determined the deficiency responsible for the sitA mutant free-living phenotypes and subsequently explored the downstream effectors that contribute to the oxidative stress sensitivity and symbiotic defect.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, phage, plasmids, and growth conditions. The bacterial strains, generalized transducing phage, and plasmids used are listed in Table 1. Escherichia coli strains were cultured at 37°C in LB. S. meliloti strains were cultured at 30°C in either LB supplemented with 2.5 mM MgSO₄ and 2.5 mM CaCl₂ (LB/MC) or M9 minimal medium with 15 mM succinate (M9) (34). M9 was prepared without iron or manganese sources in plastic containers and filter sterilized. Manganese (Mn²⁺) was added as MnSO₄ (Sigma), and iron (Fe²⁺) was added as FeSO₄ (Sigma). Plastic tubes were used for growth and sensitivity assays. Unless otherwise stated, strains were initially grown on LB/MC before dilution into M9. The following antibiotics were used: streptomycin (500 µg/ml), neomycin (200 µg/ml), chloramphenicol (20 µg/ml), and tetracycline (10 µg/ml).

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TABLE 1. Bacterial strains, phage, and plasmids

Genetic techniques and DNA manipulations. Transductions with M12 were performed as described previously (15). Random mini-Tn5-GusNm (mTn5) mutagenesis and triparental matings were performed as described previously (13, 37). The protocols of Sambrook and Russell (39) were used for routine manipulations of plasmid and chromosomal DNAs. To construct strains GWBD2, GWBD8, GWBD9, and GWBD10 a 300- to 500-bp internal fragment of sodB, pphA, degP1, or relA, respectively, was cloned into pKNOCK-Tc. To construct strains GWBD4 and GWBD11, a 300- to 500-bp internal fragment of sitB or degP2, respectively, was cloned into pJH104. The resulting plasmids were conjugated into Rm1021 via triparental mating to introduce a disruption by single-crossover homologous recombination. Single-crossover disruptions were transduced into Rm1021 and verified by PCR. To construct plasmid pGW1, full-length sitA was PCR amplified and cloned into pMSO4. pGW1 was introduced into GWBD1 by triparental mating.

Hydrogen peroxide and plumbagin sensitivity assays. For H₂O₂ sensitivity assays, S. meliloti cultures were diluted into M9 medium and grown for 3 days to an optical density at 600 nm (OD₆₀₀) of 1.0 to 1.2. These cultures were diluted to an OD₆₀₀ of 0.1 into M9 with or without 10 µM MnSO₄ or 30 µM FeSO₄ and grown for 2 h at 30°C before the addition of H₂O₂ to a final concentration of 8 mM. At the indicated times, samples were taken, serially diluted, and spotted onto LB agar. After 4 days of growth at 30°C, the number of CFU was determined. For plumbagin sensitivity assays, S. meliloti cultures were diluted into M9 medium with or without 10 µM MnSO₄ or 30 µM FeSO₄ and grown overnight to an OD₆₀₀ of 1.0 to 1.2. These cultures were diluted to an OD₆₀₀ of 0.1 into M9 with or without 10 µM MnSO₄ or 30 µM FeSO₄ before the addition of plumbagin to a final concentration of 0.75 mM. At the indicated times, samples were taken, serially diluted, and spotted onto LB agar. After 4 days of growth at 30°C, the number of CFU was determined. H₂O₂ concentrations in solution were determined using an Amplex Red hydrogen peroxide/peroxidase assay kit (Molecular Probes).

Bacterial lysates, enzyme activity assays, and immunoblots. S. meliloti lysates were obtained from cultures grown in M9 with or without 10 µM MnSO₄ or 30 µM FeSO₄. Cells were disrupted by sonication, and protein concentration was determined by the Bradford assay. In-gel catalase and superoxide dismutase assays were performed as previously described (6, 19). Immunoblotting was performed as previously described (38) with polyclonal Mn superoxide dismutase antibody (QED Biosciences).

RNA isolation and RT-PCR. Total RNA was isolated from S. meliloti cultures grown in M9 by using the QIAGEN RNeasy minikit and was quantified by OD₂₆₀. DNA contamination was tested for by PCR amplification of 23S rRNA genomic sequence in the absence of reverse transcriptase. Reverse transcription-PCR (RT-PCR) was performed using the SuperScript one-step RT-PCR kit (Invitrogen) with increasing amounts of RNA for 15 or 35 PCR cycles to determine the linear range for each target transcript. Primers were designed to amplify 300- to 500-bp internal sequences for the indicated genes. RT-PCRs with primers specific to 23S rRNA were used as a control to ensure equal amounts of RNA template between reactions.

Plant assays. Alfalfa seedlings were nodulated on petri dishes of Jensen agar as previously described (32). Three-day-old seedlings were inoculated with approximately 10⁷ bacteria. Plant height, nodule number, and nitrogen fixation were determined after 4 weeks of growth. Nitrogen fixation was quantified via the acetylene reduction assay (46) Ethylene-acetylene separation and quantitation were carried out on a Shimadzu GC-8A gas chromatograph. The amount of ethylene produced was calculated by peak integration and conversion to picomoles of ethylene formed per nodule by comparison to a standard curve developed from injected standard amounts of ethylene. Four-week-old nodules were examined by electron microscopy using standard techniques (11, 23).

Bacteria were isolated as previously described (18). Briefly, nodules were surface sterilized with 70% ethanol for 30 s, followed by three water washes, and then treatment with 10% bleach for 30 s, followed by three water washes. The nodules were then crushed with a sterile pestle in 100 µl of LB/MC containing 0.3 M glucose. The nodule suspension was serially diluted (10⁰ to 10^–6) and plated onto LB/MC containing 0.3 M glucose.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sitA::mTn5 mutant is symbiotically defective on Medicago sativa. The sitA mutant that we identified is disrupted by an mTn5 transposon at base 162 of the 903-bp sitA open reading frame. After determining the H₂O₂ sensitivity and symbiotic defect of the original isolate, we transduced the sitA::mTn5 allele into the parental wild-type strain, Rm1021. We tested several transductants and confirmed that both the H₂O₂ sensitivity and the symbiotic defect are linked to the mTn5 insertion in sitA. We selected one transductant, GWBD1, for further study. We refer to strain GWBD1 as the sitA::mTn5 mutant below.

We inoculated the sitA::mTn5 mutant and Rm1021 onto Medicago sativa seedlings. After 4 weeks, we assayed nodule number, plant height, and ability to fix nitrogen as measured by acetylene reduction (Table 2). Both sitA::mTn5 mutant- and Rm1021-inoculated plants began producing nodules after 1 week. After 4 weeks, sitA::mTn5 mutant-inoculated plants showed approximately 20% more nodules than Rm1021-inoculated plants. Nodules from Rm1021-inoculated plants were mostly pink (Fig. 1A) due to leghemoglobin, which is a marker of a healthy symbiosis (1).

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TABLE 2. Plant heights and nitrogenase activities of M. sativa plants inoculated with Rm1021 and derivative strains after 4 weeks of growth

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FIG. 1. Morphology and ultrastructure of M. sativa nodules induced by Rm1021 and the sitA::mTn5 mutant. (A) Pink nodule induced by Rm1021. (B) Small white nodule induced by the sitA::mTn5 mutant. (C) Intermediate nodule induced by the sitA::mTn5 mutant. (D) Ultrastructure of pink nodule induced by Rm1021 (bar, 1.2 µm). (E) All nodules induced by the sitA::mTn5 mutant had a similar ultrastructure. The ultrastructure of a small white nodule induced by the mutant is shown (bar, 1.5 µm). Starch granules (S) and plant vacuoles (V) are indicated.

In contrast, all sitA::mTn5 mutant-inoculated plants produced mainly small white nodules, indicative of a defective symbiosis (Fig. 1B). In addition to the small white nodules, sitA::mTn5 mutant-inoculated plants produced another type of nodule that was intermediate in size between healthy pink nodules and defective white nodules (Fig. 1C). This type of nodule has a slight pink zone at the base proximal to the root. We did observe an occasional pink nodule on a mutant-inoculated plant but at a very low frequency.

sitA::mTn5 mutant-inoculated plants were significantly shorter than and had a substantial reduction in acetylene reduction activity compared with Rm1021-inoculated plants (Table 2). These characterizations were consistent with the decrease in healthy pink nodules observed on sitA::mTn5 mutant-inoculated plants. While we were characterizing the sitA::mTn5 mutant, a report describing a sitA deletion was published (10). In agreement with our observations, that report showed that acetylene reduction was decreased for alfalfa inoculated with the sitA deletion strain, but the physiology of the symbiotic defect was not further characterized.

sitA::mTn5 mutant-inoculated plants showed reduced but measurable levels of acetylene reduction, suggesting that the strain is able to colonize the nodules. However, the majority of nodules produced by the sitA::mTn5 mutant were small and white, indicative of a failed symbiosis. This suggests that if this strain is able to colonize nodules, it does so with a greatly reduced efficiency. To gain a better understanding of the effect of the sitA transposon disruption on symbiotic development of S. meliloti, we examined the ultrastructure of each type of nodule induced by the sitA::mTn5 mutant by electron microscopy (Fig. 1E). Each nodule type from sitA::mTn5 mutant-inoculated plants had a similar ultrastructure that differed markedly from that of nodules induced by the parental strain Rm1021 (Fig. 1D). The most striking difference was the presence of large starch granules in nodules induced by the sitA::mTn5 mutant. These large deposits, found lining the wall of the plant cell in sitA::mTn5 mutant-induced nodules, were completely absent from Rm1021-induced nodules. Deposits of large starch granules have also been observed in several other symbiotically defective strains of S. meliloti, though the reason for their presence is still not understood (14, 22, 33). We also observed that the plant vacuoles in sitA::mTn5 mutant-induced nodules were much smaller and displayed more irregular shapes than vacuoles in Rm1021-induced nodules.

We found it intriguing that although the sitA::mTn5 mutant clearly produces three morphologically distinct types of nodules, the ultrastructures of the three types were very similar. This observation led us to hypothesize that although the mutant was found in each type of nodule, perhaps its intracellular survival varies. To determine the number of bacteria in the nodules, we crushed each type of nodule from sitA::mTn5 mutant-inoculated plants as well as nodules induced by Rm1021 to recover any bacteria present and plated for CFU. Interestingly, although the micrographs show comparable numbers of bacteria in each type of sitA::mTn5 mutant-induced nodule, we were able to recover bacteria only from the pink nodules. However, these nodules contained approximately 1,000-fold-fewer bacteria than pink nodules from Rm1021-inoculated plants (data not shown).

It was possible that the bacteria recovered from nodules induced by the sitA::mTn5 mutant had acquired a suppressor mutation allowing for their survival. To determine whether a suppressor had accumulated, we confirmed the presence of the mTn5 insertion in sitA in bacteria isolated from pink sitA::mTn5 mutant-induced nodules and used these isolates to reinoculate M. sativa. After 4 weeks, we found the same plant phenotypes and spectrum of nodules as were found when plants were inoculated with the original sitA::mTn5 strain (data not shown). This result indicates that the pink nodules formed by the sitA::mTn5 mutant are not due to the acquisition of a suppressing mutation. As M. sativa is an outbred tetraploid, we hypothesize that the formation of pink nodules with the sitA::mTn5 mutant is most likely due to the genetic variability of the plant host (9).

Genetic characterization of the sitA::mTn5 mutant. sitA is the first transcribed gene of the sitABCD operon (17). We therefore felt it essential to determine the effect of the mTn5 disruption on expression of the downstream genes in the operon, sitBCD. We performed RT-PCR on genes sitB, sitC, and sitD. In the sitA::mTn5 mutant, sitB, sitC, and sitD are all expressed but at reduced levels compared to those in Rm1021 (Fig. 2). Although there is decreased expression from sitBCD, we nevertheless found that expression of sitA alone from a plasmid was sufficient to rescue the sitA::mTn5 symbiotic defect (data not shown). However, sitBCD expression is still important for symbiotic development, since a strain carrying a polar disruption of sitB (GWBD4) shows a symbiotic defect equivalent to that of the sitA::mTn5 mutant (data not shown).

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FIG. 2. Transcript levels of sitB, sitC, and sitD were analyzed by RT-PCR from total RNA extracted from the Rm1021 and sitA::mTn5 strains grown in M9 medium. The transcript level of 23S rRNA was determined as a loading control.

Growth of the sitA::mTn5 mutant is limited for manganese. When we first isolated the sitA transposon mutant, we observed that it formed colonies more slowly than Rm1021 on LB plates. sitA is part of the sitABCD operon, which is designated a putative iron/manganese ABC transporter operon in the S. meliloti genome (17). It seemed likely that the decreased growth rate of the sitA::mTn5 mutant is due to insufficient uptake of one or both of these metals. We subsequently found that the strain has a decreased growth rate compared to Rm1021 in LB liquid medium and that the growth defect is even more severe in M9 medium. Rm1021 was able to grow in M9 alone; however, the sitA::mTn5 mutant showed no appreciable growth (Fig. 3A). To determine whether either iron or manganese limitation was responsible for the growth defect, we supplemented the growth medium with either MnSO₄ or FeSO₄. Addition of 10 µM MnSO₄ completely restored the growth rate of the sitA::mTn5 mutant to that of Rm1021 (Fig. 3B). Addition of FeSO₄ caused an increase in the growth rate of Rm1021 but did not affect the growth of the sitA::mTn5 mutant even when included at 30 µM (Fig. 3C). Taken together, these data indicate that the growth defect of the sitA::mTn5 mutant is due to a defect in manganese uptake and not a defect in iron uptake.

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FIG. 3. Growth of Rm1021 and the sitA::mTn5 mutant in M9 minimal medium. Strains grown on LB medium were diluted to an OD600 of 0.001 in M9, and growth was monitored by OD600. Due to detection limitations of the spectrophotometer, we could determine measurements only above an OD600 of 0.01. (A) Rm1021 () and sitA::mTn5 mutant () growth in M9. (B) Rm1021 () and sitA::mTn5 mutant () growth in M9 supplemented with 10 µM MnSO4. (C) Rm1021 () and sitA::mTn5 mutant () growth in M9 supplemented with 30 µM FeSO4.

Sensitivity of the sitA::mTn5 mutant to H₂O₂ is specifically rescued by manganese. The sitA::mTn5 mutant was identified as H₂O₂ sensitive in our initial screen (12). In that study, we assayed H₂O₂ sensitivity by using zone-of-inhibition assays on LB plates. Under those conditions, the sitA::mTn5 mutant appeared to be only slightly more sensitive than Rm1021 to H₂O₂. Similar to the case for the growth defect, we postulated that the sensitivity would be more apparent in minimal medium. As the sitA::mTn5 mutant does not grow in M9, we first grew the strain in M9 supplemented with 10 µM MnSO₄. We then diluted the culture in M9 without supplementation and allowed the cultures to grow for 3 days to starve the cells for Mn²⁺ before assaying H₂O₂ sensitivity. Indeed, the sitA::mTn5 mutant cultures starved for Mn²⁺ showed a substantial increase in sensitivity to H₂O₂ relative to Rm1021 (Fig. 4A). Furthermore, we found that if manganese-starved sitA::mTn5 mutant cultures were then supplemented with 10 µM MnSO₄ for 2 h, the increased sensitivity to H₂O₂ was greatly diminished (Fig. 4B). We did not observe increased rescue with longer incubation times or increased MnSO₄ concentrations of up to 100 µM (data not shown). Also in agreement with the growth phenotypes, supplementing the medium with FeSO₄ does not rescue the H₂O₂ sensitivity (Fig. 4C).

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FIG. 4. Sensitivity of Rm1021 and the sitA::mTn5 mutant to H2O2. Strains grown in M9 minimal medium were diluted to an OD600 of 0.1 in M9 alone or M9 supplemented with 10 µM MnSO4 or 30 µM FeSO4. After 2 h, the strains were challenged with 8 mM H2O2. Samples were taken at the indicated times postchallenge and plated for CFU. The data are represented as percent survival relative to t = 0 h. (A) Rm1021 () and sitA::mTn5 () strains in M9. (B) Rm1021 () and sitA::mTn5 () strains in M9 supplemented with 10 µM MnSO4. (C) Rm1021 () and sitA::mTn5 () strains in M9 supplemented with 30 µM FeSO4.

As discussed above, Mn²⁺ ions in low-molecular-weight complexes are able to detoxify H₂O₂. We were concerned that the rescue of H₂O₂ sensitivity by MnSO₄ might be a general detoxification of the medium by free Mn²⁺. To test this, we monitored the decomposition of H₂O₂ in M9 with and without 10 µM MnSO₄ over time by using the Amplex Red Detection system. We found that addition of 10 µM MnSO₄ does not affect the H₂O₂ concentration in solution (data not shown).

Catalases are the major component of an adaptive response to H₂O₂ and have been shown to be upregulated in bacteria compromised for other oxidative stress defenses (21, 25, 43). S. meliloti contains three catalases, KatA, KatB, and KatC, none of which are manganese dependent (21, 28, 44). We speculated that since the sitA::mTn5 mutant exhibits increased sensitivity to H₂O₂, catalase activity may be altered in this strain. We assayed total cell lysates of the sitA::mTn5 mutant and Rm1021 grown in M9, with and without MnSO₄, for catalase activity and observed an altered catalase activity profile in the mutant (Fig. 5). Most notably, KatA activity is upregulated in the sitA:mTn5 mutant. KatA is controlled by the global sensor of H₂O₂ stress, OxyR (27). Upregulation of KatA suggests that even during normal growth, the sitA::mTn5 mutant experiences an increased intracellular stress from reactive oxygen species. Furthermore, we found that the sitA::mTn5 mutant grown in M9 supplemented with MnSO₄ showed a catalase profile that appeared identical to that of Rm1021, indicating that manganese starvation is responsible for the increase in oxidative stress (Fig. 5).

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FIG. 5. Catalase activity patterns of Rm1021 and the sitA::mTn5 mutant in M9 minimal medium. Total protein lysates were isolated from saturated cultures grown in M9 supplemented or not with 10 µM MnSO4, and 35 µg from each lysate was subjected to electrophoresis through a native 7.5% polyacrylamide gel and assayed for catalase activity. The positions of KatA, KatB, and KatC according to Siguad et al. (44) are noted.

When comparing the catalase profile of the sitA::mTn5 mutant to its sensitivity to H₂O₂, we noted two intriguing phenomena. First, although KatA activity is strongly upregulated in the sitA::mTn5 mutant, the strain is still quite sensitive to H₂O₂. Second, although addition of 10 µM MnSO₄ restores the catalase activity profile of the sitA::mTn5 mutant to that of Rm1021 (Fig. 5), the mutant still remains slightly sensitive to H₂O₂ (Fig. 4B). These results suggest that there is an additional mechanism employed by S. meliloti to manage H₂O₂ stress that is dependent on SitA and possibly on full SitBCD activity as well.

The sitA::mTn5 mutant shows increased sensitivity to superoxide that is specifically rescued by manganese. Our initial characterization of the sitA::mTn5 mutant indicated an increased sensitivity to superoxide (12). This phenotype was more apparent in M9, where the mutant showed very strong sensitivity to plumbagin (Fig. 6A), a redox-cycling quinone that generates superoxide (20). The sensitivity of the sitA::mTn5 mutant to plumbagin appears to be much more pronounced than its sensitivity to H₂O₂, as it requires only overnight starvation of Mn²⁺ before sensitivity to plumbagin is observed. As with the sensitivity to H₂O₂, growing the sitA::mTn5 mutant in M9 supplemented with MnSO₄ prior to assaying with plumbagin decreased its sensitivity (Fig. 6B). Also like H₂O₂ sensitivity, supplementation with FeSO₄ did not rescue plumbagin sensitivity of the sitA::mTn5 mutant (Fig. 6C). The addition of MnSO₄ had an even greater effect on the plumbagin sensitivity of the mutant than on its H₂O₂ sensitivity, as addition of MnSO₄ to the assay completely abolished sensitivity to plumbagin.

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FIG. 6. Sensitivity of Rm1021 and the sitA::mTn5 mutant to plumbagin. Strains grown overnight in M9 supplemented or not with 10 µM MnSO4 or 30 µM FeSO4 were diluted to an OD600 of 0.1 in the same medium and challenged with 0.75 mM plumbagin. Samples were taken at the indicated times postchallenge and plated for CFU. The data are represented as percent survival relative to t = 0 h. (A) Rm1021 () and sitA::mTn5 () strains in M9. (B) Rm1021 () and sitA::mTn5 () strains in M9 supplemented with 10 µM MnSO4. (C) Rm1021 () and sitA::mTn5 () strains in M9 supplemented with 30 µM FeSO4.

SodB activity is decreased in the sitA::mTn5 mutant and correlates with a decrease in intracellular SodB level. Having established the ability of MnSO₄ to alleviate the sensitivity of the sitA::mTn5 mutant to both H₂O₂ and plumbagin, we sought to determine the mechanism(s) through which manganese was acting to provide oxidative stress protection. There is an increasing list of enzymes that require Mn²⁺ for activity (26). S. meliloti encodes a superoxide dismutase (SodB) that can use either Fe²⁺ or Mn²⁺ as a cofactor but shows a much higher activity when utilizing Mn²⁺ (40). In-gel activity assays using total cell lysates from M9 cultures showed that SodB activity is greatly reduced in the sitA::mTn5 mutant (Fig. 7A). The decrease in activity parallels a similar decrease in SodB protein level from the same lysate (Fig. 7B). Addition of MnSO₄ to the cell lysate does not rescue superoxide dismutase activity (data not shown). However, when cell lysates were made from sitA::mTn5 mutant cultures grown in M9 supplemented with 10 µM MnSO₄, both SodB activity and protein levels were restored nearly to wild-type levels (Fig. 7A and B). This rescue is specific, as addition of FeSO₄, even at 30 µM, to sitA::mTn5 mutant cultures did not restore SodB activity (data not shown).

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FIG. 7. Superoxide dismutase activity patterns and protein profiles of Rm1021 and the sitA::mTn5 mutant in M9 minimal medium. (A and B) Total protein lysates were isolated from saturated cultures grown in M9 supplemented or not with 10 µM MnSO4, and 35 µg of each lysate was subjected to electrophoresis and either stained for superoxide dismutase activity (A) or blotted for SodB protein (B). A sodB mutant strain (GWBD2) is shown as a negative control for the absence of SodB. (C) The transcript level of sodB was analyzed by RT-PCR from total RNA extracted from Rm1021 and sitA::mTn5 strains grown in M9 medium. The transcript level of 23S rRNA was determined as a loading control.

To understand at which level of expression Mn²⁺ affects SodB, we performed RT-PCR using mRNA extracted from Rm1021 and sitA::mTn5 mutant cultures grown in M9. Using primers specific to a 300-bp internal fragment of the sodB gene, we found that transcription of sodB is unaffected in the sitA::mTn5 mutant under these conditions (Fig. 7C). In conjunction with the Western blot showing a substantial decrease in SodB levels, this implies that the Mn²⁺ is affecting the production of SodB by enhancing translation of sodB mRNA and/or stabilizing SodB once translated.

Our biochemical data implicate SodB in the oxidative stress sensitivity of the sitA::mTn5 mutant. We constructed a sodB mutant (GWBD2) and a sitA::mTn5 sodB double mutant (GWBD3) to test for epistasis of oxidative stress sensitivity. Unfortunately, we were unable to find conditions under which we could accurately compare the plumbagin sensitivities of the sitA::mTn5 and sodB mutants due to the >10⁶-fold increase in plumbagin sensitivity of the sodB mutant relative to the sitA::mTn5 mutant. However, we did find that the decrease in SodB was not responsible for sitA::mTn5 mutant H₂O₂ sensitivity, as the sodB mutant did not show increased sensitivity to H₂O₂ compared to Rm1021 (data not shown).

The decrease in SodB activity is not the cause of the sitA::mTn5 symbiotic defect. The genetic and biochemical data offered strong evidence that the plumbagin sensitivity of the sitA::mTn5 mutant is due to a decrease in SodB. To determine whether a loss of SodB activity could also be responsible for the symbiotic defect, we inoculated alfalfa with our sodB mutant. After 4 weeks, the plant height and acetylene reduction activity were measured (Table 2). We found the sodB mutant-inoculated plants to be indistinguishable from Rm1021-inoculated plants, discounting the decrease in SodB activity as the cause of the sitA::mTn5 symbiotic defect. In agreement with this result, we also found that plants inoculated with our sitA::mTn5 sodB double mutant exhibited the same degree of symbiotic deficiency as sitA::mTn5 mutant-inoculated plants alone (Table 2).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The recognized roles of manganese in bacterial physiology are steadily growing (31). Proper uptake of manganese through SitABCD transporter homologs of S. enterica serovar Typhimurium and S. pyogenes has been shown to be required for full virulence of these pathogens (7, 29). We have shown that in S. meliloti, sitA is involved in Mn²⁺ uptake and a disruption of SitA results in a symbiotic defect, thus extending the requirement of manganese to bacterium-plant symbiosis as well.

We found that a polar disruption of sitB causes a symbiotic defect similar to that of sitA::mTn5, thereby implicating sitB, and potentially sitCD as well, as being required for proper symbiotic development. A previous study that isolated a sitB transposon mutant found that disruption of sitB did not affect symbiosis (35). That study used both a different strain of S. meliloti (strain 242) and different cultivar of Medicago sativa (Creola), which may explain the discrepancy between their results and ours. However, in agreement with this previous study, we found that our sitB mutant (GWBD4) had a severe growth defect in medium lacking manganese. As with the sitA::mTn5 mutant, our sitB mutant did not grow in M9 and this defect was fully complemented by the addition of 10 µM MnSO₄ (data not shown). As our sitA::mTn5 and sitB mutants share very similar phenotypes both in planta and in free-living states, we feel that the phenotypes we observe in the sitA::mTn5 mutant represent a defect in the entire SitABCD transporter, which requires all of its components to function efficiently.

Our work demonstrates that SitABCD plays a very important role in Mn²⁺ transport in S. meliloti and that it is a deficiency in Mn²⁺ that is responsible for the oxidative stress sensitivity and growth defect observed from disrupting sitA in Rm1021. Our findings are consistent with studies of SitABCD homologs in S. enterica serovar Typhimurium, which showed that SitABCD can transport both Fe²⁺ and Mn²⁺ but that transport of Mn²⁺ is favored 100 times over that of Fe²⁺ (30). Previous work suggested that Mn²⁺ alone could not rescue the growth defect of a sitA deletion strain (10). The authors of that work graciously sent us their sitA deletion strain for comparison with our sitA::mTn5 strain. We found that the discrepancy in the results is due to the concentration of MnSO₄ added to the medium. The previous authors reported that 1 µM MnSO₄ was not sufficient to rescue the sitA deletion strain growth defect. We also found that 1 µM MnSO₄ was not sufficient to rescue the growth defect of our sitA::mTn5 strain but that adding 10 µM MnSO₄ rescues the growth phenotype of both our and their sitA mutant strains when tested under our conditions (data not shown).

Our work has established the important role for manganese in oxidative stress protection in S. meliloti. The requirement for manganese in this capacity is most likely much greater than what we have observed. Since manganese is needed for growth, we were required to provide a low level of manganese to serve this function. Therefore, we are unable to observe the actual severity of oxidative stress sensitivity in a truly manganese-free S. meliloti culture.

Our biochemical analysis strongly suggests that the superoxide sensitivity of the sitA::mTn5 mutant is due to a decrease in SodB activity and abundance. S. meliloti SodB has previously been shown to be able to utilize either iron or manganese but has stronger activity in the presence of manganese (40). Given the dual metal utilization of SodB, we found it interesting that disruption of manganese uptake specifically causes a decrease in SodB activity and protein level. Our results suggest a strong role for manganese, but not iron, for SodB to carry out its physiological role.

Our work has shown that the transcription of sodB is not affected in the sitA::mTn5 background. This indicates that Mn²⁺ regulates SodB at the translational or posttranslational level. One possibility is that Mn²⁺ is required for translation of the sodB transcript, possibly by binding to the transcript and altering its secondary structure in a riboswitch-type manner (48). Alternatively, Mn²⁺ insertion in SodB may be required for enzyme stability so that in the absence of Mn²⁺, SodB is rapidly degraded.

Superoxide is dismutated by superoxide dismutase into O₂ and H₂O₂, the latter of which is decomposed by catalase to O₂ and water (20). Given the decrease in SodB activity observed in the sitA::mTn5 mutant, the 10,000-fold increase in sensitivity to plumbagin is understandable (Fig. 6A). We found that a sodB mutant does not show increased sensitivity to H₂O₂, ruling out SodB deficiency as the cause of the H₂O₂ sensitivity of the sitA::mTn5 mutant. Since SodB functions upstream of hydrogen peroxide detoxification, it is not unexpected that the sodB mutant was not sensitive to hydrogen peroxide.

Rm1021 does not appear to encode a Mn²⁺-dependent catalase or any other obvious Mn²⁺-dependent enzyme that could detoxify H₂O₂ (17), so what is the SitA-dependent mechanism responsible for the H₂O₂ sensitivity observed in the sitA::mTn5 mutant? One explanation is that intracellular manganese alone acts as an oxidative stress defense mechanism. In nonprotein low-molecular-weight complexes, Mn²⁺ has been shown to decompose H₂O₂ (24, 45). Supporting this idea are the results that the sensitivity to H₂O₂ of the sitA::mTn5 mutant could be alleviated by growing this strain with MnSO₄ for only 2 h after starvation, while protection against plumbagin required a much longer growth period in the presence of MnSO₄. This may be because time is required for de novo synthesis of SodB after addition of Mn²⁺, whereas once taken up, Mn²⁺ ions alone are able to attenuate the toxicity of H₂O₂, decreasing the time required for rescue. Complicating this, however, is the observation that the addition of exogenous Mn²⁺ does not fully restore resistance of the sitA::mTn5 mutant to H₂O₂ (Fig. 4B). This may be because once the mutant is depleted of manganese, its uptake of Mn²⁺ is not adequate to fully restore sufficient intracellular levels required for H₂O₂ resistance.

The sitA::mTn5 mutant is symbiotically defective. Although a sodB mutant of Rm5000 has previously been reported to be symbiotically deficient, we find that a sodB mutant in the Rm1021 background does not exhibit a detectable symbiotic defect (41). This discrepancy may be due to differences in strain background. Our findings eliminate the decrease in SodB level in the sitA::mTn5 mutant as the cause of the symbiotic defect. Thus, the question still remains: what causes the symbiotic defect in the sitA::mTn5 mutant? From homology searches, potential Mn²⁺-dependent enzymes encoded in the S. meliloti genome include DegP1, DegP2, PphA, RelA/SpoT, and SodB. We have created disruptions of each of these genes by using single-crossover suicide plasmids and have found that only the relA/spoT mutant shows a symbiotic defect, a result that was shown previously (47). The relA/spoT mutant and the sitA::mTn5 mutant do share some free-living phenotypes, such as an inability to grow in M9; however, the sitA::mTn5 mutant does not possess other characteristics of the relA/spoT mutant, such as overproduction of succinoglycan (data not shown). We are continuing to investigate this relationship. Although we postulate that the sitA::mTn5 symbiotic defect is due to loss of an Mn²⁺-dependent enzymatic activity, it may be that S. meliloti is part of an increasing list of bacteria that utilize the activities of Mn²⁺ alone and that it is simply a decrease in intracellular Mn²⁺ that is the cause of the symbiotic defect in the sitA::mTn5 mutant.

 
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 by 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.

 
* 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.

Published ahead of print on 15 December 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, March 2007, p. 2101-2109, Vol. 189, No. 5
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