Dataset Information

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans.

ABSTRACT: ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn(2+) or Mn(2+) specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn(2+) over Mn(2+) specificity, the Zn(2+) ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn(2+)-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn(2+)-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn(2+)-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn(2+) specificity.

SUBMITTER: Handali M

PROVIDER: S-EPMC4424328 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans.

Handali Melody M Neupane Durga P DP Roychowdhury Hridindu H Yukl Erik T ET

The Journal of biological chemistry 20150318 19

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attrib ...[more]

PMID: 25787075

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Project description:Paracoccus denitrificans is a valuable model organism due to its versatile respiration capability and bioenergetic flexibility, both of which are critical to its survival in different environments. Quorum sensing (QS) plays a crucial role in the regulation of many cell functions; however, whether QS systems play a role in P. denitrificans is unknown. In this study, we demonstrated that iron uptake systems in P. denitrificans were directly regulated by a newly identified QS system. Genes coding for TonB-dependent systems, which transport chelated iron, were transcribed at higher levels in the QS-defective mutants. In contrast, genes coding for the Fbp system, which is TonB independent and transports unchelated ferric iron, were downregulated in the mutants. In brief, QS in P. denitrificans triggers a switch in iron uptake from TonB-dependent to TonB-independent transport during biofilm formation as higher concentrations of iron accumulate in the exopolysaccharide (EPS). Switching from TonB-dependent iron uptake systems to TonB-independent systems not only prevents cells from absorbing excess iron but also conserves energy. Our data suggest that iron uptake strategies are directly regulated by QS in Paracoccus denitrificans to support their survival in available ecological niches.IMPORTANCE As iron is an important trace metal for most organisms, its absorption is highly regulated. Fur has been reported as a prevalent regulator of iron acquisition. In addition, there is a relationship between QS and iron acquisition in pathogenic microbes. However, there have been few studies on the iron uptake strategies of nonpathogenic bacteria. In this study, we demonstrated that iron uptake systems in Paracoccus denitrificans PD1222 were regulated by a newly identified PdeR/PdeI QS system during biofilm formation, and we put forward a hypothesis that QS-dependent iron uptake systems benefit the stability of biofilms. This report elaborates the correlation among QS, iron uptake, and biofilm formation and thus contributes to an understanding of the ecological behavior of environmental bacteria.

Project description:Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins.

Dataset Information

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans.

Publications

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans.

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