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Appl Environ Microbiol. Oct 2008; 74(20): 6473–6475.
Published online Aug 29, 2008. doi:  10.1128/AEM.01590-08
PMCID: PMC2570276

ShmR Is Essential for Utilization of Heme as a Nutritional Iron Source in Sinorhizobium meliloti[down-pointing small open triangle]

Abstract

The bacterium Sinorhizobium meliloti is able to use heme as a nutritional iron source. Here, we show that the iron-regulated shmR gene encodes an outer membrane protein required for growth on heme. Furthermore, an shmR mutant is resistant to the toxic heme analog gallium protoporphyrin. Thus, the receptor protein of the heme transport system has been identified in S. meliloti.

Heme is an iron protoporphyrin, which serves as the prosthetic group of heme proteins. Many bacteria, including Sinorhizobium meliloti and other rhizobia, can use heme as a nutritional iron source (8). In gram-negative bacteria, a heme-binding outer membrane receptor is a component of a heme transport system. Heme uptake systems in Rhizobium leguminosarum (14) and Bradyrhizobium japonicum (7) have been described, but a heme receptor was identified only in the latter species. Bioinformatic analysis of the S. meliloti 1021 genome identified two putative heme receptor genes, smc02726 and smc04205. We previously characterized the Smc02726 homolog in S. meliloti 242. This protein was shown to bind heme; thus, it was named ShmR (Sinorhizobium heme receptor) (2). In the S. meliloti 1021 genome, the shmR gene is on the chromosome and is not clustered within an operon. In this study, we investigate the role of ShmR in heme utilization in S. meliloti 1021.

In order to assess differential expression of outer membrane proteins in response to iron, cells were grown in M3 (2) iron-replete media or in media where iron was chelated with ethylenediamine-di-o-hydroxyphenylacetic acid (EDDHA). Outer membrane fractions were prepared as previously described by Battistoni et al. (2), which includes a step to solubilize inner membrane proteins and thus separate them from the insoluble outer membrane proteins. Analysis of the outer membrane protein profile of the S. meliloti 1021 cells revealed the presence of two proteins, 82 and 91 kDa in size, that were absent in iron-replete cultures. These two proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as RhtA and ShmR, respectively (data not shown). ShmR migrated more slowly than its predicted size of ca. 80 kDa, which is not unusual for membrane proteins. RhtA is an outer membrane receptor for the siderophore rhizobactin, which is specifically expressed under iron limitation (6). We did not attempt to identify other outer membrane proteins.

To further address the function of shmR, we constructed a mutant strain disrupted in that gene as follows. A 2.7-kb DNA fragment containing the shmR gene was amplified from the S. meliloti 1021 genome by PCR using primers 5′-ATTCGTCTCGCTCCGTAAAA and 5′-CAAATTGTGCTGAAACTGAGG as the forward and reverse primers, respectively, and cloned in the EcoRV site of pBluescript II SK (Stratagene). The shmR gene was disrupted by introducing the lacZ-Gmr cassette from plasmid pAB2001 (3) into the SphI site of shmR, which creates a transcriptional shmR::lacZ fusion (Fig. (Fig.1).1). An EcoRI fragment containing the disrupted shmR gene was subcloned in the EcoRI site of pK18mobsacB (11) and mobilized into S. meliloti strain 1021 by triparental mating using DH5α(pRK2013) as a helper strain (5). Double recombinants were initially identified based on streptomycin, gentamicin, and sucrose resistance and confirmed by Southern blotting using the 2.7-kb PCR fragment as a probe.

FIG. 1.
Physical map of the S. meliloti 1021 DNA region containing the shmR gene. The trpE gene encodes an anthranilate synthase homolog; 02727 encodes a hypothetical protein. Black dots indicate the factor rho-independent transcriptional terminator. F and R ...

We compared the outer membrane profiles of the wild type with the shmR mutant strain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Battistoni et al. (2). A protein band of about 91 kDa, corresponding to ShmR according to mass spectrometry data, was present in the wild-type strain but not in the shmR mutant strain grown in iron-limited cultures (Fig. (Fig.22).

FIG. 2.
Outer membrane protein profile. Wild-type S. meliloti 1021 and shmR mutant (ShmR) strains were grown in M3 minimal medium containing 500 μM EDDHA. The arrow indicates the protein band corresponding to ShmR. MM, molecular mass markers. ...

shmR expression responds to exogenous hemin.

High-affinity heme transport systems are induced under iron limitation as a strategy to acquire iron from heme compounds (13). Previously we reported the iron responsiveness of the shmR promoter using a plasmid-borne transcriptional fusion with gfpUV (9), and similar results were obtained with a chromosomal lacZ fusion in this work (data not shown). Here, we examined the responsiveness of the shmR promoter to heme as an iron source using the plasmid-borne transcriptional fusion with gfpUV (Fig. (Fig.3).3). Under low-iron conditions with no exogenous heme, shmR promoter activity was induced, but activity decreased with increasing heme concentrations. Thus, expression of shmR is responsive to exogenous hemin and qualitatively similar to the response to iron (9).

FIG. 3.
In vivo effect of hemin concentration on shmR promoter activity in S. meliloti 1021. Wild-type strains containing either a plasmid with the presumptive shmR promoter fused to gfpUV [1021(pShm)] (9) or the pOT1 plasmid [1021(pOT)] (1) were grown in M3 ...

The S. meliloti shmR mutant is resistant to the toxic heme analog Ga-PPIX.

Bacteria that can transport heme into cells are sensitive to killing by the heme analogue gallium protoporphyrin (Ga-PPIX) (12). To test whether ShmR is required for internalization of heme, we examined the effects of Ga-PPIX on growth inhibition of the wild-type and shmR mutant strains. Ga-PPIX was spotted into wells of iron-chelated solid-medium plates containing cells, and the Ga-PPIX effect was scored as an inhibition halo around the wells. Growth of wild-type strain S. meliloti 1021 was affected by Ga-PPIX, as shown by an inhibition halo around the well indicating that Ga-PPIX enters cells. However, growth of the shmR mutant was not inhibited, showing that ShmR is required for internalization of the heme analogue. When the strains were grown in iron-sufficient solid media, the presence of Ga-PPIX did not inhibit bacterial growth (data not shown), which is consistent with iron-dependent regulation of shmR.

The shmR gene is required for utilization of heme as a nutritional iron source.

To investigate the effect of a mutation in the S. meliloti shmR gene on heme-mediated iron nutrition, we tested the ability of the shmR mutant to use different iron sources for growth. Under iron limitation, S. meliloti synthesizes and transports the siderophore rhizobactin 1021 as an iron scavenging system (6). The expression of this system may interfere with the ability to establish whether the shmR strain can use heme as an iron source. To circumvent this problem, we carried out the studies using the rhizobactin-deficient strain H38 (10). This strain is disrupted in the rhrA gene, which encodes an activator of the rhizobactin 1021 system (6). This mutant does not produce the endogenous siderophore but retains the ability to utilize heme compounds and heterologous siderophores (10). As a consequence, the rhrA mutant is unable to grow in iron-restricted medium unless a nutritional iron source other than ferric-rhizobactin 1021 is added to the medium. The shmR gene was disrupted in strain H38 as described above to construct an rhrA shmR double mutant. When cultured in iron-replete M3 medium, the rhrA mutant and rhrA shmR double mutant exhibited similar growth properties. By contrast, media supplemented with the iron chelator EDDHA did not support growth of either strain. However, the addition of hemin to the iron-chelated medium restored growth of the rhrA mutant but not of the rhrA shmR double mutant (Fig. (Fig.4).4). These data show that shmR is essential for the utilization of heme as a sole iron source by S. meliloti.

FIG. 4.
The shmR gene is required to use heme as an iron source. S. meliloti H38 strain (black) and its shmR derivative mutant (white) were grown in M3 minimal medium supplemented with either 200 μM EDDHA (triangles), 37 μM FeCl3 (circles), or ...

S. meliloti strain 1021 is able to use heme proteins, as well as heme, as sources of iron (8). However, the shmR strain was unable to grow on hemoglobin or leghemoglobin but retained the ability to grow on FeCl3, ferrichrome, and ferric-rhizobactin 1021 (Table (Table1).1). These results demonstrate that the shmR gene is necessary for iron acquisition from heme, hemoglobin, and leghemoglobin and that this gene encodes the only functional heme receptor present under the conditions tested.

TABLE 1.
Effect of shmR gene mutation on the ability to use different compounds as sole iron sources

ShmR is not essential for nitrogen fixation.

Two independent plant assays were carried out using the wild-type and shmR strains to inoculate 20 germinated seedlings in nitrogen-free Jensen medium as previously described (9). No significant differences could be detected in plant dry weight or visualization of nodules formed between Medicago sativa cv. Creola plants inoculated with wild-type or shmR mutant strains (data not shown). These results indicate that the ShmR protein is not essential for symbiosis or nitrogen fixation in alfalfa in the condition assayed here.

Conclusions.

The results presented herein demonstrate that the outer membrane heme-binding protein ShmR plays an essential role in heme internalization and iron nutrition from heme, hemoglobin, and leghemoglobin in the free-living form of S. meliloti. Collectively, our findings show that ShmR is the only functional heme receptor in S. meliloti in free-living cells.

Acknowledgments

This research was supported by a grant from the NIH Fogarty International Research Collaboration, award R03 TW007353, to M.R.O., with E.F. as the foreign collaborator; by NIH grant GM067966 to M.R.O.; and by a grant from PEDECIBA-Uruguay to E.F.

We thank Federico Battistoni for mass spectrometry assays.

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 August 2008.

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