Identification of an Iron-Regulated, Hemin-Binding Outer Membrane Protein inSinorhizobium meliloti

Abstract

Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport.

The Neisseria meningitidis ZnuD Zinc Receptor Contributes to Interactions with Epithelial Cells and Supports Heme Utilization when Expressed in Escherichia coli

“…Binding was performed in high-ionicstrength conditions to prevent nonspecific ionic interaction with the matrix, and multiple extensive washings in the presence of N-laurylsarcosine were performed to further remove nonspecific interaction prior to the elution of bound proteins. Analogously to other heme receptors demonstrated to interact with heme agarose in vitro (3,15), ZnuD was retained on heme resins (Fig. 2B), while no nonspecific protein binding to heme agarose was detected in the noninduced sample.…”

“…Strains and plasmids used in this study are listed in Table 1. Meningococcal strains were grown with 5% CO 2 at 37°C with GC base agar (Difco) supplemented with 0.4% glucose and 0.68 mM Fe(NO 3 ) 3 or in GC broth with the same supplements and 0.043% NaHCO 3 or RPMI without phenol red and supplemental with 0.3 mg/ml glutamine. BHI medium (37 g/liter brain heart infusion) with 1.25% fetal bovine serum (FBS) was used when kanamycin selection was required.…”

“…5B), overlapped with this putative promoter and, consequently, could repress znuD expression through interference with RNA polymerase binding. As many receptors involved in the acquisition of various iron sources are regulated by Fur (39), we checked for the presence of Fur box sequences in the znuD promoter region using the meningococcal Fur box consensus motif, (natwat) 3 (17). A putative Fur box with four mismatches was revealed on both the coding and noncoding strands to locate upstream and adjacent to the putative Zur binding site (Fig.…”

Role of the Regulatory GenerirAin the Transcriptional Response ofSinorhizobium melilotito Iron Limitation

“…The measurements were obtained with a LightCycler instrument (Roche, Mannheim, Germany). The rhbA and shmR genes were chosen since previous reports showed that there was iron-responsive regulation of these genes (3,34). Iron-dependent regulation of the hmuS genes was confirmed by glucuronidase assays (data not shown).…”

“…A number of siderophores have been characterized in different rhizobia; these include the trihydroxamate vicibactin of Rhizobium leguminosarum (10,11), the amino polycarboxylic acid siderophore of Sinorhizobium meliloti DM4 (59), and the citrate-based dihydroxamate rhizobactin 1021 produced by S. meliloti 1021 (46). In addition to their own or exogenous ferri-siderophores, rhizobia are also known to use a wide range of other iron sources, including ferric citrate, heme, and hemoglobin (3,11,39,71). This flexibility with regard to iron utilization probably reflects adaptation to complex environmental conditions and underlines the importance of iron acquisition for rhizobia.…”