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The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen
Author(s) -
Kehres David G.,
Zaharik Michelle L.,
Finlay B. Brett,
Maguire Michael E.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01922.x
Subject(s) - escherichia coli , biology , salmonella enterica , biochemistry , divalent , enterobacteriaceae , bacteria , superoxide dismutase , reactive oxygen species , microbiology and biotechnology , enzyme , gene , chemistry , genetics , organic chemistry
NRAMPs (natural resistance‐associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe 2+ and Mn 2+ , cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium , and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia . The genes are designated mntH because the two enterobacterial NRAMPs encode H + ‐stimulated, highly selective manganese(II) transport systems, accounting for all Mn 2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the K m for 54 Mn 2+ (≈ 0.1 µM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg 2+ and Ca 2+ did not inhibit 54 Mn 2+ uptake. Ni 2+ , Cu 2+ and Zn 2+ inhibited uptake with K i s greater than 100 µM, Co 2+ with a K i of 20 µM and Fe 2+ with a K i that decreased from 100 µM at pH 7.6 to 10 µM at pH 5.5. Fe 3+ and Pb 2+ inhibited weakly, exhibiting K i s of 50 µM, while Cd 2+ was a potent inhibitor with a K i of about 1 µM. E. coli MntH had a similar inhibition profile, except that K i s were three‐ to 10‐fold higher. Both S. typhimurium and E. coli MntH also transport 55 Fe 2+ however, the K m s are equivalent to the K i s for Fe 2+ inhibition of Mn 2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli , mntH :: lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn 2+ and Cd 2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH :: lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn 2+ transporter MntH and Mn 2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.