ShmR Is Essential for Utilization of Heme as a Nutritional Iron Source in Sinorhizobium meliloti

The bacterium Sinorhizobium meliloti is able to use heme asa nutritional iron source. Here, we show that the iron-regulatedshmR gene encodes an outer membrane protein required for growthon heme. Furthermore, an shmR mutant is resistant to the toxicheme analog gallium protoporphyrin. Thus, the receptor proteinof the heme transport system has been identified in S. meliloti.

INTRODUCTION

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INTRODUCTION

Heme is an iron protoporphyrin, which serves as the prostheticgroup of heme proteins. Many bacteria, including Sinorhizobiummeliloti and other rhizobia, can use heme as a nutritional ironsource (8). In gram-negative bacteria, a heme-binding outermembrane receptor is a component of a heme transport system.Heme uptake systems in Rhizobium leguminosarum (14) and Bradyrhizobiumjaponicum (7) have been described, but a heme receptor was identifiedonly 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 Smc02726homolog in S. meliloti 242. This protein was shown to bind heme;thus, it was named ShmR (Sinorhizobium heme receptor) (2). Inthe S. meliloti 1021 genome, the shmR gene is on the chromosomeand is not clustered within an operon. In this study, we investigatethe role of ShmR in heme utilization in S. meliloti 1021.

In order to assess differential expression of outer membraneproteins in response to iron, cells were grown in M3 (2) iron-repletemedia or in media where iron was chelated with ethylenediamine-di-o-hydroxyphenylaceticacid (EDDHA). Outer membrane fractions were prepared as previouslydescribed by Battistoni et al. (2), which includes a step tosolubilize inner membrane proteins and thus separate them fromthe insoluble outer membrane proteins. Analysis of the outermembrane protein profile of the S. meliloti 1021 cells revealedthe presence of two proteins, 82 and 91 kDa in size, that wereabsent in iron-replete cultures. These two proteins were identifiedby matrix-assisted laser desorption ionization-time of flightmass spectrometry as RhtA and ShmR, respectively (data not shown).ShmR migrated more slowly than its predicted size of ca. 80kDa, which is not unusual for membrane proteins. RhtA is anouter membrane receptor for the siderophore rhizobactin, whichis specifically expressed under iron limitation (6). We didnot attempt to identify other outer membrane proteins.

To further address the function of shmR, we constructed a mutantstrain disrupted in that gene as follows. A 2.7-kb DNA fragmentcontaining the shmR gene was amplified from the S. meliloti1021 genome by PCR using primers 5'-ATTCGTCTCGCTCCGTAAAA and5'-CAAATTGTGCTGAAACTGAGG as the forward and reverse primers,respectively, and cloned in the EcoRV site of pBluescript IISK (Stratagene). The shmR gene was disrupted by introducingthe lacZ-Gm^r cassette from plasmid pAB2001 (3) into the SphIsite of shmR, which creates a transcriptional shmR::lacZ fusion(Fig. 1). An EcoRI fragment containing the disrupted shmR genewas subcloned in the EcoRI site of pK18mobsacB (11) and mobilizedinto S. meliloti strain 1021 by triparental mating using DH5 ${alpha}$ (pRK2013)as a helper strain (5). Double recombinants were initially identifiedbased on streptomycin, gentamicin, and sucrose resistance andconfirmed by Southern blotting using the 2.7-kb PCR fragmentas a probe.

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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 indicate forward and reverse primers, respectively. S, SphI restriction site where the lacZ-Gm^r reporter was inserted; E, EcoRI restriction site used for subcloning the shmR::lacZ-Gm^r transcriptional fusion into the suicide plasmid.

We compared the outer membrane profiles of the wild type withthe shmR mutant strain by sodium dodecyl sulfate-polyacrylamidegel electrophoresis as described by Battistoni et al. (2). Aprotein band of about 91 kDa, corresponding to ShmR accordingto mass spectrometry data, was present in the wild-type strainbut not in the shmR mutant strain grown in iron-limited cultures(Fig. 2).

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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.

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shmR expression responds to...

High-affinity heme transport systems are induced under ironlimitation as a strategy to acquire iron from heme compounds(13). Previously we reported the iron responsiveness of theshmR promoter using a plasmid-borne transcriptional fusion withgfpUV (9), and similar results were obtained with a chromosomallacZ fusion in this work (data not shown). Here, we examinedthe responsiveness of the shmR promoter to heme as an iron sourceusing the plasmid-borne transcriptional fusion with gfpUV (Fig.3). Under low-iron conditions with no exogenous heme, shmR promoteractivity was induced, but activity decreased with increasingheme concentrations. Thus, expression of shmR is responsiveto exogenous hemin and qualitatively similar to the responseto iron (9).

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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 minimal medium supplemented with 150 µM EDDHA and different hemin concentrations. 1021(pOT) was used as a control. Relative fluorescence, in arbitrary fluorescence units per unit of optical density at 620 nm (OD620nm), was determined. The data shown are the mean values of four independent experiments done by triplicate. The error bars indicate one standard deviation.

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

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The S. meliloti shmR...

Bacteria that can transport heme into cells are sensitive tokilling by the heme analogue gallium protoporphyrin (Ga-PPIX)(12). To test whether ShmR is required for internalization ofheme, we examined the effects of Ga-PPIX on growth inhibitionof the wild-type and shmR mutant strains. Ga-PPIX was spottedinto wells of iron-chelated solid-medium plates containing cells,and the Ga-PPIX effect was scored as an inhibition halo aroundthe wells. Growth of wild-type strain S. meliloti 1021 was affectedby Ga-PPIX, as shown by an inhibition halo around the well indicatingthat Ga-PPIX enters cells. However, growth of the shmR mutantwas not inhibited, showing that ShmR is required for internalizationof the heme analogue. When the strains were grown in iron-sufficientsolid media, the presence of Ga-PPIX did not inhibit bacterialgrowth (data not shown), which is consistent with iron-dependentregulation of shmR.

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

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The shmR gene is...

To investigate the effect of a mutation in the S. meliloti shmRgene on heme-mediated iron nutrition, we tested the abilityof the shmR mutant to use different iron sources for growth.Under iron limitation, S. meliloti synthesizes and transportsthe siderophore rhizobactin 1021 as an iron scavenging system(6). The expression of this system may interfere with the abilityto establish whether the shmR strain can use heme as an ironsource. To circumvent this problem, we carried out the studiesusing the rhizobactin-deficient strain H38 (10). This strainis disrupted in the rhrA gene, which encodes an activator ofthe rhizobactin 1021 system (6). This mutant does not producethe endogenous siderophore but retains the ability to utilizeheme compounds and heterologous siderophores (10). As a consequence,the rhrA mutant is unable to grow in iron-restricted mediumunless a nutritional iron source other than ferric-rhizobactin1021 is added to the medium. The shmR gene was disrupted instrain H38 as described above to construct an rhrA shmR doublemutant. When cultured in iron-replete M3 medium, the rhrA mutantand rhrA shmR double mutant exhibited similar growth properties.By contrast, media supplemented with the iron chelator EDDHAdid not support growth of either strain. However, the additionof hemin to the iron-chelated medium restored growth of therhrA mutant but not of the rhrA shmR double mutant (Fig. 4).These data show that shmR is essential for the utilization ofheme as a sole iron source by S. meliloti.

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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 200 µM EDDHA plus 10 µM hemin (squares). OD600nm, optical density at 600 nm.

S. meliloti strain 1021 is able to use heme proteins, as wellas heme, as sources of iron (8). However, the shmR strain wasunable to grow on hemoglobin or leghemoglobin but retained theability to grow on FeCl₃, ferrichrome, and ferric-rhizobactin1021 (Table 1). These results demonstrate that the shmR geneis necessary for iron acquisition from heme, hemoglobin, andleghemoglobin and that this gene encodes the only functionalheme receptor present under the conditions tested.

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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.

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ShmR is not essential...

Two independent plant assays were carried out using the wild-typeand shmR strains to inoculate 20 germinated seedlings in nitrogen-freeJensen medium as previously described (9). No significant differencescould be detected in plant dry weight or visualization of nodulesformed between Medicago sativa cv. Creola plants inoculatedwith wild-type or shmR mutant strains (data not shown). Theseresults indicate that the ShmR protein is not essential forsymbiosis or nitrogen fixation in alfalfa in the condition assayedhere.

Conclusions.

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Conclusions.

The results presented herein demonstrate that the outer membraneheme-binding protein ShmR plays an essential role in heme internalizationand iron nutrition from heme, hemoglobin, and leghemoglobinin the free-living form of S. meliloti. Collectively, our findingsshow that ShmR is the only functional heme receptor in S. melilotiin free-living cells.

ACKNOWLEDGMENTS

This research was supported by a grant from the NIH FogartyInternational Research Collaboration, award R03 TW007353, toM.R.O., with E.F. as the foreign collaborator; by NIH grantGM067966 to M.R.O.; and by a grant from PEDECIBA-Uruguay toE.F.

We thank Federico Battistoni for mass spectrometry assays.

FOOTNOTES

* Corresponding author. Mailing address: Laboratorio de Ecología Microbiana, IIBCE, Av. Italia 3318, Montevideo 11600, Uruguay. Phone: 598 (2) 4871616, ext. 146. Fax: 598 (2) 4875548. E-mail: efabiano{at}iibce.edu.uy

Published ahead of print on 29 August 2008.

REFERENCES

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