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Journal of Bacteriology, March 2009, p. 1519-1527, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01661-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Essential Role for the BacA Protein in the Uptake of a Truncated Eukaryotic Peptide in Sinorhizobium meliloti{triangledown}

Victoria L. Marlow,1 Andreas F. Haag,1 Hajime Kobayashi,2 Vivien Fletcher,1 Marco Scocchi,3 Graham C. Walker,2 and Gail P. Ferguson1*

School of Medicine & Dentistry, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom,1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,2 Department of Life Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy3

Received 25 November 2008/ Accepted 4 December 2008


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ABSTRACT
 
The inner membrane BacA protein is essential for the establishment of chronic intracellular infections by Sinorhizobium meliloti and Brucella abortus within plant and mammalian hosts, respectively. In their free-living state, S. meliloti and B. abortus mutants lacking BacA have reductions in their outer membrane lipid A very-long-chain fatty acid (VLCFA) contents and exhibit low-level resistance to the glycopeptide bleomycin in comparison to their respective parent strains. In this paper we investigate the hypothesis that BacA is involved in peptide uptake in S. meliloti. We determined that an S. meliloti {Delta}bacA mutant is completely resistant to a truncated form of the eukaryotic peptide Bac7, Bac7(1-16), and this phenotype appears to be independent of its lipid A alteration. Subsequently, we discovered that BacA and/or Escherichia coli SbmA is essential for fluorescently labeled Bac7(1-16) uptake in S. meliloti. Given that there are hundreds of root nodule-specific peptides within the legume host, our data suggest that BacA-mediated peptide uptake could play a central role in the chronic infection process of S. meliloti. However, since we determined that two symbiotically defective S. meliloti bacA site-directed mutants (with the Q193G and R389G mutations, respectively) with known reductions in their lipid A VLCFA contents are still capable of peptide uptake, these findings suggest that BacA-dependent peptide uptake cannot fully account for the essential role of BacA in the legume symbiosis. Further, they provide evidence that the BacA function that leads to the S. meliloti lipid A VLCFA modification plays a key role in the chronic infection of legumes.


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INTRODUCTION
 
The alphaproteobacterium Sinorhizobium meliloti can be found either free-living in the soil or in a symbiotic relationship with leguminous plants such as alfalfa. The symbiosis is initiated by the legume, which releases flavonoids into the soil when grown under conditions of nitrogen limitation (33). The flavonoids then induce the production of nodulation (Nod) factors in S. meliloti, which signal a series of events within the legume root, including root hair curling (for reviews, see references 4 and 16). Bacterially synthesized exopolysaccharides are then required for the formation of plant-derived structures known as infection threads (3, 36). S. meliloti multiplies and travels down the infection threads until it is eventually released into membrane-bound compartments within the plant cell known as symbiosomes (30). Within the symbiosome, S. meliloti differentiates into an elongated, nitrogen-fixing bacteroid (for recent reviews, see references 17 and 20). Although this interaction is regarded as a beneficial symbiosis because the plant obtains a nitrogen source from S. meliloti bacteroids and the bacterium gains carbon from the plant, it can also be viewed as a chronic intracellular infection, since S. meliloti bacteroids persist for extensive periods of time within the plant host (24).

16S rRNA comparisons followed by genome sequencing revealed that S. meliloti strain Rm1021 is closely related to Brucella species (6, 15, 35), which are chronic intracellular mammalian pathogens. Brucella species cause abortions in animals and brucellosis, a severe and debilitating disease, in humans (5). Although animal vaccines exist, there is currently no human vaccine against Brucella species. This is a serious issue, because Brucella species are highly infectious potential bioterrorism/biowarfare agents (14). Despite the very different outcomes of brucellae and S. meliloti for their respective hosts, parallels can be drawn, since they both need to survive intracellularly within membrane-bound, acidic compartments for the long term (30, 35, 38).

The BacA protein has been found to be specifically required for the development of S. meliloti Rm1021 bacteroids (18). Unlike the S. meliloti parent strain, which establishes a chronic intracellular infection in alfalfa, S. meliloti mutants lacking BacA are unable to differentiate into bacteroids and die shortly after their entry into the plant cell. Likewise, a Brucella abortus mutant lacking BacA is able to invade macrophages and initiate acute infections in BALB/c mice but is defective in establishing chronic infections (24). BacA is predicted to be an integral inner membrane protein. In both S. meliloti and B. abortus, BacA is necessary for the synthesis of normal lipid A, a component of the lipopolysaccharide, which forms the outermost layer of the outer membrane of gram-negative bacterial cells. For both free-living S. meliloti and B. abortus, loss of BacA function results in a 50% reduction in the amount of lipid A that is modified with 27-OH C28:0 and 29-OH C30:0 very-long-chain fatty acids (VLCFAs) (9, 12). Based on the limited sequence similarity between BacA and the human adrenoleukodystrophy protein, which is thought to be involved in the transport of activated VLCFA out of the cytoplasm into the peroxisome, we proposed that BacA could be involved in the transport of activated VLCFAs out of the cytoplasm, where they could then be used to modify the lipid A in the outer membrane (9).

In addition to affecting the structure of lipid A, S. meliloti and B. abortus bacA-null mutants display low-level resistance to the glycopeptide bleomycin (12, 19, 40). Although the mechanism by which BacA confers increased sensitivity to bleomycin on S. meliloti is unknown, we determined that it is independent of its effect on lipid A modification (11). In Escherichia coli, loss of the BacA homolog SbmA also confers increased sensitivity to a number of peptides, such as bleomycin, microcins, and truncated forms of Bac7 [Bac7(1-16) and Bac7(1-35)] (19, 23, 28). In the case of microcin B17 and the Bac7 peptides, it was found that the SbmA protein is involved in their uptake in E. coli (23, 28). Transcriptome analysis of the S. meliloti legume host Medicago truncatula has revealed that hundreds of cysteine-rich secreted peptides are produced within the root nodules (1, 31), and these peptides have been hypothesized to play a role in S. meliloti bacteroid development (32). Combined, these findings led us to investigate the hypothesis that the BacA protein could be involved in peptide uptake in S. meliloti. However, since the sequences of the root nodule-specific cysteine-rich peptides are highly variable (1, 31) and the synthesis of cysteine-rich peptides is extremely difficult (21), we used a truncated version of the eukaryotic Bac7 peptide [Bac7(1-16)] and a fluorescently labeled form (28, 42) to investigate peptide uptake in S. meliloti. Given that the full-length Bac7 peptide was originally isolated from bovine leukocytes (13) and B. abortus is a bovine pathogen (5), this study could also provide valuable insights into the potential role of the BacA protein in chronic mammalian pathogens.

We found that in contrast to E. coli, where loss of SbmA reduces but does not eliminate Bac7 uptake (28), BacA is essential for the uptake of Bac7(1-16) in S. meliloti. Therefore, this study has revealed an essential function for BacA in peptide uptake in S. meliloti. Furthermore, our analysis of Bac7(1-16) uptake in a series of previously constructed S. meliloti BacA site-directed mutants (25), taken together with our previous analyses (9, 25), suggests that the function of BacA in lipid A VLCFA modification plays a critical role in the chronic infection of legumes.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. All bacterial strains and plasmids used in this study are described in Table 1. The S. meliloti strains used are all derivatives of the sequenced strain Rm1021 (15). For all experiments, S. meliloti strains were grown either in Luria-Bertani broth (LB) alone (41) or in LB supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LB-MC) for 48 h. When required, antibiotics were used at the following concentrations (in micrograms per milliliter): streptomycin (Sm), 500; neomycin (Nm), 200; tetracycline (Tc), 5.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Bac7-derived peptides. The N-terminal fragment comprising amino acids 1 to 16 of Bac7 was synthesized as previously described (2). A fluorescently labeled version of Bac7(1-16), Bac71-16-BY, was prepared by the linkage of the thiol-reactive dye BODIPY FL N-(2-aminethyl)maleimide (Invitrogen) to an additional C-terminal cysteine residue as reported previously (42). Peptides with purity greater than 95% were dissolved in slightly acidified double-distilled water; their concentrations were determined as previously reported (2); and they were stored at –20°C until use.

Bac7 sensitivity assays. Mid-exponential-phase cultures of the indicated strains were harvested, washed, and diluted to an optical density at 600 nm (OD600) of 0.05 in fresh LB medium. After the addition of either Bac7(1-16) or Bac71-16-BY to the indicated concentrations, the cultures were incubated at 30°C. At the indicated times, samples were removed and serially diluted in LB medium, and then 10-µl aliquots were plated in triplicate onto LB agar plates. CFUs were calculated after 72 h at 30°C. All data sets shown are representative of at least two independent experiments where similar trends were observed. The mean CFU per milliliter in the three 10-µl aliquots was plotted, and the error bars represent the standard deviations from the means. Where indicated, the significance of viability differences between strains were assessed by the Student unpaired t test using Microsoft Excel 2003.

Bac71-16-BY uptake assays. Uptake assays were performed as described previously for E. coli with modifications (28). Mid-exponential-phase cultures of the indicated strains were harvested, washed, and resuspended to an OD600 of 0.05 in fresh LB medium. After the addition of Bac71-16-BY, the cultures were incubated at 30°C for 1 h. At the indicated times, cells were washed in fresh LB medium to remove extracellular Bac71-16-BY and were then resuspended in 50 mM sodium phosphate buffer (pH 7.0). To account for extracellular binding of Bac71-16-BY, the cultures were then either left untreated or treated with 1 mg ml–1 of the extracellular quencher of fluorescence trypan blue (TB) for 10 min at room temperature. A Becton Dickinson LSR II flow cytometer equipped with a 488-nm laser was used to measure the fluorescence parameter of single cells after treatment. BODIPY FL maleimide (505/513 nm) fluorescence was measured using a 530/30-nm band-pass filter (FL1). All parameters were set on the logarithmic scale, and 10,000 events were collected for each analysis. Data were acquired and analyzed using Becton Dickinson FACSDiva and FlowJo (Tree Star Inc.) software, respectively. To further confirm that Bac71-16-BY was entering into the cytoplasm of S. meliloti, cultures were treated as for the flow cytometry experiments except that they were resuspended in LB to an OD600 of ~0.8 to 1.0 and then exposed to 0.5 µM Bac71-16-BY. After a wash, cultures were analyzed by fluorescence microscopy (Zeiss Axiostar Plus) using the fluorescein isothiocyanate filter set.


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RESULTS
 
BacA and the E. coli SbmA protein sensitize S. meliloti to Bac7(1-16). A previous study demonstrated that the BacA homolog SbmA increases the sensitivity of E. coli to truncated forms of the eukaryotic peptide Bac7 (28). Since the S. meliloti BacA protein is 79% similar (64% identical) to the E. coli SbmA protein (19), we investigated whether the BacA protein was also involved in sensitizing S. meliloti to the truncated Bac7 peptide Bac7(1-16) (RRIRPRPPRLPRPRPR). We determined that treatment of the S. meliloti strain Rm1021 with 0.25 µM Bac7(1-16) resulted in loss of cell viability, whereas in the absence of BacA, S. meliloti remained viable over the time course of the experiment (Fig. 1A). Interestingly, the S. meliloti bacA mutant also remained resistant to exposure to higher doses (1 µM) of Bac7(1-16) over the same time course (Fig. 1B). These data provide evidence that the BacA protein sensitizes S. meliloti to Bac7(1-16)-induced cell death.


Figure 1
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  		FIG. 1. BacA and SbmA sensitize S. meliloti to Bac7(1-16) and Bac71-16-BY. (A and B) Cultures of the indicated strains were exposed to either 0.25 µM (A) or 1 µM (B) Bac7(1-16), and cell viability was determined before (open bars) and 1 h after (shaded bars) addition. The significant values (***, P < 0.001) represent comparisons of the Rm1021 {Delta}bacAsp mutant to the parent strain and of the Rm1021 {Delta}bacAsp mutant with either pJG51A (the S. meliloti bacA gene cloned into pRK404) or pAI351 (the E. coli sbmA gene cloned into pRK404) to the same mutant with pRK404 (control plasmid with no insert). (C) Cultures of the Rm1021 {Delta}bacAsp (open squares), Rm1021 acpXL::pk18mobGII (open triangles), and Rm1021 {Delta}bacAsp/acpXL::pk18mobGII (filled triangles) mutant strains and of the parent strain, Rm1021 (filled squares), were treated with 0.25 µM Bac7(1-16), and cell viability was determined at the indicated times. (D) Same as panel A except that Bac71-16-BY was used.

To confirm that the sensitivity of S. meliloti to Bac7(1-16) was due to BacA, the S. meliloti Rm1021 bacA-null mutant carrying either pRK404 (control vector) or pJG51A (S. meliloti wild-type bacA gene cloned into pRK404) (18) was exposed to Bac7(1-16), and cell viability was monitored (Fig. 1A and B). We determined that the presence of pJG51A, but not the control plasmid, sensitized the S. meliloti bacA-null mutant to Bac7(1-16)-induced cell death. Reciprocally, we determined that the E. coli SbmA protein restored the sensitivity of S. meliloti to Bac7(1-16)-induced cell death to an extent similar to that of the S. meliloti BacA protein (Fig. 1A and B). Therefore, these data show that the presence of either BacA or SbmA is essential for S. meliloti to be sensitized to Bac7(1-16)-mediated cell death.

BacA-mediated sensitivity of S. meliloti to Bac7(1-16) is independent of the lipid A VLFCA modification. We determined previously that an S. meliloti Rm1021 bacA-null mutant has a 50% reduction in its lipid A VLCFA content (9). To test whether a reduction in the outer membrane lipid A VLCFA content could account for the resistance of the S. meliloti bacA-null mutant to Bac7(1-16), we also assessed the sensitivity of an S. meliloti acpXL insertional mutant to Bac7(1-16) (Fig. 1C). AcpXL encodes a specialized acyl carrier protein that is essential for the biosynthesis of the lipid A VLCFA in free-living S. meliloti; consequently, the S. meliloti Rm1021 acpXL mutant completely lacks the lipid A VLCFA modification (10). However, despite lacking the lipid A VLCFA modification, the S. meliloti acpXL mutant had a Bac7(1-16) sensitivity identical to that of the parent strain (Fig. 1C). This provides evidence that the reduction in the outer membrane lipid A VLCFA content of the S. meliloti bacA mutant is not responsible for the Bac7(1-16) resistance phenotype. Additionally, the fact that S. meliloti Rm1021 acpXL and Rm1021 acpXL bacA-null mutants both completely lack the lipid A VLCFA modification in their free-living states (10), yet loss of BacA still confers resistance to Bac7(1-16) in the acpXL mutant background, provides further evidence that BacA-mediated sensitivity to Bac7(1-16) is independent of the effect of BacA on lipid A.

The BacA protein is essential for the uptake of fluorescently labeled Bac71-16-BY. To investigate the hypothesis that BacA is involved in the uptake of Bac7(1-16) in S. meliloti, we used Bac7(1-16) labeled with the fluorescent dye BODIPY (Bac71-16-BY) (28). As was the case for unlabeled Bac7(1-16) (Fig. 1A to C), we observed that S. meliloti possessing either the S. meliloti bacA gene or the E. coli sbmA gene was highly sensitive to killing by Bac71-16-BY, whereas S. meliloti lacking either BacA or SbmA was resistant (Fig. 1D). Since the fluorescent labeling of Bac7(1-16) did not interfere with BacA-mediated sensitivity to this peptide in S. meliloti, we used Bac71-16-BY to monitor Bac7(1-16) uptake. Using flow cytometry, we observed that incubation of S. meliloti Rm1021 with Bac71-16-BY results in dramatically increased fluorescence of cells relative to cells of the untreated control culture (Fig. 2A). However, although the S. meliloti cells were washed extensively prior to the flow cytometry analysis to remove any surface-attached Bac71-16-BY, we could not be sure that some of the increased fluorescence was not due to extracellular binding. To rule this out, S. meliloti cells were also treated with the extracellular quencher of fluorescence TB (28) prior to flow cytometry analysis (Fig. 2B). Our observation that treatment with TB had only a slight effect on the fluorescence profile of the parent strain in the presence of Bac71-16-BY confirms that the increased fluorescence is due to the intracellular accumulation of this peptide.


Figure 2
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  		FIG. 2. The BacA protein is essential for the uptake of Bac71-16-BY in S. meliloti. Cultures of the indicated strains were either left untreated (empty histograms) or treated with 0.25 µM Bac71-16-BY (shaded histograms) for 1 h. The cultures were then treated with (+) or without (–) TB preincubation as indicated and were analyzed by flow cytometry using a 530/30-nm band-pass filter to measure BODIPY FL (505/513) fluorescence. Logarithmic signal amplification was used.

To determine whether the BacA protein was involved in the uptake of Bac71-16-BY into S. meliloti, we also assessed the fluorescence of an Rm1021 bacA-null mutant before and after exposure to Bac71-16-BY by flow cytometry (Fig. 2C). In contrast to the results for the parent strain (Fig. 2A), after incubation with Bac71-16-BY (Fig. 2C) we observed a much smaller increase in the fluorescence of cells of the S. meliloti bacA-null mutant, and that increase was abolished by TB treatment (Fig. 2D), indicating that it was due to extracellular binding. Possibly the reduction in the lipid A VLCFA content (9, 12) in the S. meliloti bacA-null mutant could cause a change in the properties of the outer membrane that increases the amount of extracellular binding of Bac71-16-BY in the absence of TB. However, since there was no increased fluorescence in the presence of TB, these data show that, in contrast to the situation in E. coli, where SbmA contributes partially to the uptake of truncated forms of Bac7 (28), BacA appears to be playing an essential role in the intracellular accumulation of Bac71-16-BY in S. meliloti.

For E. coli, it was shown previously, using antibodies to Bac7(1-35) in combination with immunogold electron microscopy, that this peptide enters into the cytoplasm (37). To further confirm that Bac71-16-BY was entering into the cytoplasm of S. meliloti cells in a BacA-dependent manner, we also analyzed cells by fluorescent microscopy (data not shown). Consistent with our flow cytometry analysis, we determined that cells of the parent strain were highly fluorescent after Bac71-16-BY treatment, whereas we detected virtually no fluorescence in cells of the S. meliloti bacA-null mutant under the same conditions. Since the fluorescence in the parent strain was uniformly distributed, this provides evidence that Bac71-16-BY is entering into cells rather than accumulating in the periplasmic space.

BacA or E. coli SbmA complements the Bac71-16-BY uptake defect of the S. meliloti bacA-null mutant. To confirm that the differences we observed by flow cytometry (Fig. 2) were due to the BacA protein, we also assessed the uptake of Bac71-16-BY into an Rm1021 bacA-null mutant carrying either a control plasmid (pRK404) (Fig. 3A and B) or its S. meliloti bacA+ derivative pJG51A (18) (Fig. 3C and D). As seen for the Rm1021 bacA-null mutant alone, we observed some increased fluorescence of Rm1021 with the control plasmid after Bac71-16-BY addition (Fig. 3A), but this was eliminated by TB incubation (Fig. 3B). In contrast, we observed a dramatic increase in the fluorescence of the Rm1021 bacA-null mutant with pJG51A after incubation with Bac71-16-BY, both with and without subsequent incubation with TB (Fig. 3D and C, respectively). Additionally, the cloned S. meliloti bacA gene also restored Bac71-16-BY-induced killing in the S. meliloti bacA-null mutant (Fig. 1D). Taken together, these results confirm that BacA is essential for the uptake and subsequent killing of S. meliloti by Bac71-16-BY.


Figure 3
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  		FIG. 3. BacA or E. coli SbmA complements the Bac71-16-BY uptake defect of the S. meliloti bacA-null mutant. Cultures of the Rm1021 {Delta}bacAsp mutant with the indicated plasmid were either left untreated (empty histograms) or treated with 0.25 µM Bac71-16-BY (shaded histograms) for 1 h. The cultures were then treated with (+) or without (–) TB preincubation as indicated and were analyzed by flow cytometry as described in the legend to Fig. 2.

Since we had shown earlier that the E. coli SbmA protein could compensate for the absence of BacA in sensitizing S. meliloti to Bac7(1-16), we assessed whether the E. coli SbmA protein could also compensate for the role of BacA in Bac71-16-BY uptake (Fig. 3E and F). Our analysis showed that the E. coli SbmA protein could restore the ability of S. meliloti lacking BacA to accumulate Bac71-16-BY. Furthermore, the E. coli SbmA protein also restored the sensitivity of the S. meliloti bacA-null mutant to Bac71-16-BY-induced cell death (Fig. 1D).

Site-directed mutations in the bacA gene affect the sensitivity of S. meliloti to Bac7(1-16) and the uptake of Bac71-16-BY. As mentioned above, peptides are produced both by the root nodules of legumes and by mammalian cells (1, 13, 22, 31). Therefore, BacA-dependent peptide uptake could be playing a critical role in the chronic infection process of S. meliloti and/or B. abortus. However, in this case, the peptide(s) would not result in cell death but instead could play a role in either the establishment or the maintenance (or both) of the chronic infection. To gain insights into whether BacA-dependent peptide uptake was solely responsible for its essential role in chronic intracellular infections, we took advantage of a set of previously constructed plasmid-borne S. meliloti bacA site-directed mutants in which conserved residues had been mutated to glycines (25). Four of the amino acids changed in this set of mutants are conserved with the human X-linked adrenoleukodystrophy protein (9). We previously found that the S. meliloti missense bacA mutants (the Q193G, D198G, R284G, and R389G mutants), all of which are defective in chronic intracellular infections of alfalfa (25), have reductions in their lipid A VLCFA contents relative to that of S. meliloti with the wild-type bacA gene (9).

We determined that seven of nine of the symbiotically defective S. meliloti bacA site-directed mutants, including the D198G and R284G mutants, were highly resistant to Bac7(1-16)-induced cell death (Fig. 4A), suggesting that these mutants are defective in peptide uptake. In contrast, the symbiotically defective BacA Q193G and R389G site-directed mutants remained sensitive to the toxic effects of Bac7(1-16) (Fig. 4A), suggesting that they were capable of peptide uptake. We therefore analyzed the abilities of four of these symbiotically defective site-directed mutants (the Q193G, D198G, R284G, and R389G mutants) to take up Bac71-16-BY (Fig. 4B). Consistent with the viability data (Fig. 4A), the D198G and R284G mutants were completely defective in Bac71-16-BY uptake, whereas the Q193G and R389G mutants were able to accumulate Bac71-16-BY (Fig. 4B). The fact that the Q193G and R389G mutants are still capable of peptide uptake, despite being symbiotically defective, suggests that BacA-dependent peptide uptake cannot solely account for the essential role of BacA in the legume symbiosis. Given that we found previously that the Q193G and R389G mutants have reductions in their lipid A VLCFA contents (9), these findings suggest that the loss of the BacA function necessary for lipid A VLCFA modification may account for their symbiotic defects. In contrast to the symbiotically defective S. meliloti bacA site-directed mutants, we observed that all the previously characterized symbiotically competent S. meliloti bacA site-directed mutants (25) were sensitive to Bac7(1-16)-induced killing (Fig. 5A) and/or were able to accumulate Bac71-16-BY (Fig. 5B).


Figure 4
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  		FIG. 4. S. meliloti strains with symbiotically defective site-directed mutations in the bacA gene are sensitized to Bac7(1-16) and can accumulate Bac71-16-BY. (A) Cultures of Rm8654 (Rm8002 {Delta}bacAsp mutant) with pRK404 (control), pJG51A (wild-type S. meliloti bacA gene), or the indicated symbiotically defective bacA site-directed mutant (25) were exposed to 1 µM Bac7(1-16), and cell viability was determined before (open bars) and 1 h after (shaded bars) addition. The significant values (***, P < 0.001) represent comparisons of the Rm8002 {Delta}bacAsp mutant strain with either Q193G or R389G to the Rm8002 {Delta}bacAsp mutant with pRK404 (control vector). Where indicated, these mutants were previously shown to have reductions in their lipid A VLCFA contents (9) (B) Same as panel A, except that the indicated cultures were either left untreated (open profiles) or treated with 0.25 µM Bac71-16-BY (shaded profiles) for 1 h and then analyzed by flow cytometry as described in the legend to Fig. 2. In all cases, the profiles shown were determined after preincubation with TB.


Figure 5
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  		FIG. 5. S. meliloti strains with symbiotically competent site-directed mutations in the bacA gene are sensitized to Bac7(1-16) and can accumulate Bac71-16-BY. (A) Cultures of Rm8654 (Rm8002 {Delta}bacAsp mutant) with either pRK404 (control), pJG51A (S. meliloti wild-type bacA gene), or the indicated symbiotically competent bacA site-directed mutant (25) were exposed to 1 µM Bac7(1-16), and cell viability was determined before (open bars) and 1 h after (shaded bars) addition. (B) Same as panel A, except that the indicated cultures were either left untreated (open profiles) or treated with 0.25 µM Bac71-16-BY (shaded profiles) for 1 h and then analyzed by flow cytometry as described in the legend to Fig. 2. In all cases, the profiles shown were determined after preincubation with TB.


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DISCUSSION
 
Our data demonstrate that the BacA protein plays an essential role in the uptake of a truncated form of the eukaryotic peptide Bac7 [Bac7(1-16)] into the cytoplasm of S. meliloti. Although S. meliloti and B. abortus bacA mutants have reductions in their outer membrane lipid A VLCFA modifications (9, 12), our findings indicate that BacA-dependent uptake of Bac7(1-16) does not require the lipid A VLCFA modification and that it is a separate function from the involvement of BacA in lipid A modification. First, an S. meliloti acpXL mutant, which completely lacks lipid A VLCFA modification (10), has a level of Bac7(1-16) sensitivity identical to that of the parent strain. Second, unlike S. meliloti lipid A, E. coli lipid A lacks VLCFA modifications (39), yet the E. coli SbmA protein is involved in Bac7(1-16) uptake in both E. coli (28) and S. meliloti. Additionally, we determined that the resistance of the S. meliloti bacA-null mutant to Bac7(1-16) was not due to the reduction in its lipid A VLCFA modification, since deletion of bacA in an S. meliloti acpXL mutant background still confers resistance to Bac7(1-16) compared to an S. meliloti acpXL single mutant, despite the fact that the S. meliloti acpXL single mutant and the acpXL bacA double mutant have identical lipid A alterations (10). Furthermore, the fact that two of the S. meliloti bacA site-directed mutants (the Q193G and R389G mutants), which have reductions in their lipid A VLCFA contents (9), lose viability after Bac7(1-16) treatment provides further support for the contention that the resistance of the S. meliloti bacA-null mutant to Bac7(1-16) is not an indirect consequence of the lipid A VLCFA reduction.

By further characterizing symbiotically defective S. meliloti bacA mutants (25), we discovered two mutants (the Q193G and R389G mutants) that were known to be defective in their ability to modify their lipid A species with the VLCFA (9) but were proficient in peptide uptake. The fact that these mutants were capable of peptide uptake yet symbiotically defective suggests that BacA-dependent peptide uptake cannot solely account for the essential role of this protein in S. meliloti legume infections. Furthermore, these findings provide further evidence that the function of BacA that affects the lipid A VLCFA modification plays an essential role in the legume symbiosis. Previous studies have shown that S. meliloti acpXL and lpxXL insertional mutants, which completely lack the lipid A VLCFA modification in their free-living states, are less competitive in the alfalfa symbiosis than the parent strain (10, 43), suggesting that the S. meliloti lipid A VLCFA modification plays an important but nonessential role in legume symbiosis. However, a Rhizobium leguminosarum acpXL mutant lacks the lipid A VLCFA modification in its free-living state, but passage through peas partially restores this modification (44), suggesting that there could be an additional legume-induced lipid A VLCFA modification system, which could partially compensate for the absence of AcpXL. Further studies will be necessary to determine if this is also the case for S. meliloti, but these data provide preliminary evidence that the partial reduction in the lipid A VLCFA content of the S. meliloti bacA-null mutant could affect the legume interaction. Interestingly, we determined that the BacA homolog SbmA in Salmonella enterica serovar Typhimurium is encoded in an operon with yaiW, which encodes a lipoprotein (K. Tan and G. P. Ferguson, unpublished data). Therefore, one possibility is that BacA may also affect the lipid modification of another cell envelope component in S. meliloti and that, combined with the lipid A modification, this may play a key role in the chronic infection process.

It has been hypothesized previously that root nodule peptides may play a key role in S. meliloti bacteroid development (32). Therefore, our finding that BacA plays an essential role in peptide uptake in S. meliloti, combined with the fact that all the symbiotically proficient S. meliloti bacA site-directed mutants are capable of Bac7(1-16) uptake, suggests that BacA-mediated peptide uptake could play a critical role in the chronic infection of legumes. Further biochemical analysis of the lipid A species in the remaining symbiotically defective S. meliloti bacA site-directed mutants, which are defective in peptide uptake, may provide valuable insights into the importance of peptide uptake. However, while BacA-mediated Bac7(1-16) uptake results in cell death in S. meliloti, the uptake of a nonlethal eukaryotic peptide(s) within the legume host may play an essential role in the establishment and/or maintenance of the chronic infection. As mentioned above, more than 300 cysteine-rich peptides are thought to be produced within the root nodules of legumes (1, 31, 32). These cysteine-rich peptides are 50 to 60 amino acids long and have little sequence homology but have either four or six regularly spaced cysteine residues. Therefore, the BacA-dependent uptake of one or more of these peptides could be essential for the chronic infection of legumes.

In addition to cysteine-rich peptides, two M. truncatula ENOD40 genes are also thought to encode peptides (45). One of the conserved open reading frames of ENOD40-1 is predicted to encode a 13-amino-acid peptide (MKLLCWEKSIHGS), and RNA interference-mediated silencing showed that the M. truncatula ENOD40-1 and ENOD40-2 genes are involved in bacteroid development. However, further studies will be necessary to determine whether one or more of the predicted peptides encoded by the ENDO40 genes are responsible for the biological activity. It is interesting to consider that BacA-mediated peptide uptake may play a key role in this process.

Although the Bac7(1-16) peptide was used essentially as a model peptide in this study to enable us to the test the hypothesis that BacA is a peptide transporter, the full-length 60-amino-acid Bac7 peptide is similar in length to the cysteine-rich peptides and is produced by bovine leukocytes (13). Since B. abortus is a bovine pathogen, BacA-mediated Bac7 uptake within the bovine host could be biologically relevant. Despite the fact that the truncated Bac7(1-16) peptide is toxic to S. meliloti, the full-length Bac7 peptide could be taken up into B. abortus in a BacA-dependent manner and could signal the transition into the chronic state. It has been shown previously that, in contrast to the situation in BALB/c mice (24), a B. abortus BacA-deficient mutant is not attenuated in C57BL/6 mice (34). These two types of mice are known to have differences in their immune responses (34), and consequently it is interesting to speculate that they may also have differences in the types of peptides produced. Therefore, BacA-mediated peptide uptake may be crucial for the chronic infection of B. abortus in BALB/c but not in C57BL/6 mice.

Although it is not known if the root nodules of legumes produce proline-rich peptides with structural similarities to Bac7, transcripts of proline-rich proteins are known to be upregulated (26, 46). In M. truncatula, the proline-rich protein family member MtPRP4 is predicted to have a 527-amino-acid domain composed solely of repetitive proline-rich decapeptide motifs (PPVEKPPVHK and PPVEKPPVYK) (46). These proline-rich proteins are thought to be components of the plant cell wall, and it has been proposed that they could play a role in remodeling of the host extracellular matrix following infection by S. meliloti. MtPRP4 expression is induced early in nodule development, and therefore the specific cleavage of proline-rich proteins could generate a source of proline-rich peptides, which could affect S. meliloti bacteroid development.

Currently, we do not know if BacA is directly or indirectly involved in Bac7(1-16) uptake in S. meliloti. Based on the similarity of BacA to the human adrenoleukodystrophy peroxisomal protein, we proposed a model whereby the S. meliloti and B. abortus BacA proteins could be involved in the transport of activated VLCFAs out of the cytoplasm across the inner membrane, where they could then be used to modify the lipid A in the outer membrane (9). If BacA plays a direct role in Bac7(1-16) uptake, either it can transport two very different substrates in opposite directions or our hypothesis that BacA transports an activated VLCFA is incorrect, and it may affect the lipid VLCFA modification indirectly, by a yet unknown transport system. Alternatively, it is also a formal possibility that BacA may be acting indirectly by affecting the activity of another inner membrane transporter. However, future cross-linking studies with Bac7(1-16) and/or selections for other potential S. meliloti Bac7(1-16)-resistant mutants could help distinguish between these two possibilities.

In summary, we show for the first time an essential role for the S. meliloti BacA protein in peptide uptake. Future studies using peptides from legume and mammalian hosts will provide valuable insights into the structural requirements for BacA-mediated peptide uptake and their precise effect(s) on the chronic infection processes of both S. meliloti and B. abortus. It is noteworthy that a BacA-like protein (Rv1819c) has also recently been determined to be involved in the maintenance of chronic murine infections with Mycobacterium tuberculosis (8). Since deletion of the M. tuberculosis bacA-like gene also results in increased resistance to bleomycin relative to that of the parent strain, this finding suggests that BacA-mediated peptide uptake may also play a key role in latent tuberculosis infections, which affect more than one-third of the world's population.


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ACKNOWLEDGMENTS
 
V.L.M. was supported by a BBSRC Ph.D. studentship through the University of Edinburgh, and the University of Aberdeen also helped to fund her research. This work was supported by a BBSRC grant (BB/D000564/1), an MRC New Investigator grant (G0501107) awarded to G.P.F., National Institutes of Health grant GM31010 to G.C.W., and NIEHS grant P30 ES002109 to the MIT Center for Environmental Health Sciences. University of Aberdeen startup funds also provided support for this research. G.C.W. is an American Cancer Society Research Professor.

Thanks also go to Janet Liversidge, Linda Duncan, and Raif Yucel for their training and very helpful discussion regarding flow cytometry analysis.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom. Phone: 44 1224 559147. Fax: 44 1224 555766. E-mail: g.ferguson{at}abdn.ac.uk Back

{triangledown} Published ahead of print on 12 December 2008. Back


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Journal of Bacteriology, March 2009, p. 1519-1527, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01661-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Haag, A. F., Wehmeier, S., Beck, S., Marlow, V. L., Fletcher, V., James, E. K., Ferguson, G. P. (2009). The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa. J. Bacteriol. 191: 4681-4686 [Abstract] [Full Text]  

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