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The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila.

ABSTRACT: The Gram-negative bacterium Legionella pneumophila elaborates the siderophore legiobactin. We previously showed that cytoplasmic LbtA helps mediate legiobactin synthesis, inner-membrane LbtB promotes export of legiobactin, and outer-membrane LbtU acts as the ferrisiderophore receptor. RT-PCR analyses now identified lbtC as an iron-repressed gene that is the final gene in an operon containing lbtA and lbtB. In silico analysis predicted that LbtC is an inner-membrane protein that belongs to the major facilitator superfamily (MFS). Although capable of normal growth in standard media, lbtC mutants were defective for growth on iron-depleted agar media. While producing normal levels of legiobactin, lbtC mutants were unable to utilize supplied legiobactin to stimulate growth on iron-depleted media and displayed an impaired ability to take up radiolabelled iron. All lbtC mutant phenotypes were complemented by reintroduction of an intact copy of lbtC. When a cloned copy of both lbtC and lbtU was introduced into a heterologous bacterium (Legionella longbeachae), the organism acquired the ability to utilize legiobactin to grow better on low-iron media. Together, these data indicate that LbtC is involved in the uptake of legiobactin, and based upon its predicted location is most likely the mediator of ferrilegiobactin transport across the inner membrane. The data are also a unique documentation of how an MFS protein can promote bacterial iron-siderophore import, standing in contrast to the vast majority of studies which have defined ABC-type permeases as the mediators of siderophore import across the Gram-negative inner membrane or the Gram-positive cytoplasmic membrane.

SUBMITTER: Chatfield CH

PROVIDER: S-EPMC3352117 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila.

Chatfield Christa H CH Mulhern Brendan J BJ Viswanathan V K VK Cianciotto Nicholas P NP

Microbiology (Reading, England) 20111208 Pt 3

The Gram-negative bacterium Legionella pneumophila elaborates the siderophore legiobactin. We previously showed that cytoplasmic LbtA helps mediate legiobactin synthesis, inner-membrane LbtB promotes export of legiobactin, and outer-membrane LbtU acts as the ferrisiderophore receptor. RT-PCR analyses now identified lbtC as an iron-repressed gene that is the final gene in an operon containing lbtA and lbtB. In silico analysis predicted that LbtC is an inner-membrane protein that belongs to the ma ...[more]

PMID: 22160401

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Project description:CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

Dataset Information

The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila.

Publications

The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila.

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