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Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling.

ABSTRACT: Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models. The protein interacts specifically with DNA at binding sites designated as 'Fur boxes'. In the present study, we have investigated the interaction between Fur from the fish pathogen Aliivibrio salmonicida (AsFur) and its target DNA using a combination of biochemical and in silico methods. A series of target DNA oligomers were designed based on analyses of Fur boxes from other species, and affinities assessed using electrophoretic mobility shift assay. Binding strengths were interpreted in the context of homology models of AsFur to gain molecular-level insight into binding specificity.

SUBMITTER: Berg K

PROVIDER: S-EPMC7536154 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling.

Berg Kristel K Pedersen Hege Lynum HL Leiros Ingar I

Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 20200709 4-5

Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models. T ...[more]

PMID: 32648080

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Project description:In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s).IMPORTANCE Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. While fur null mutants of many bacteria have been obtained, Fur appears to be essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P. aeruginosa cells by conditional mutagenesis and showed that Fur is dispensable for planktonic growth, while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality of Fur in P. aeruginosa and highlights unique properties of the Fur regulon in this species.

Project description:UnlabelledClostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified a C. difficile homologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated a fur insertion mutant of C. difficile. To identify the genes regulated by Fur in C. difficile, we used microarray analysis to compare transcriptional differences between the fur mutant and the wild type when grown in high-iron medium. The fur mutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated in C. difficile. Among those Fur- and iron-repressed genes were C. difficile genes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased during C. difficile infection using the hamster model. Our data suggest that C. difficile expresses multiple iron transport mechanisms in response iron depletion in vitro and in vivo.ImportanceClostridium difficile is the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an "urgent" antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here that C. difficile regulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditions in vitro and during C. difficile infection of the host. This suggests that during a C. difficile infection, iron availability is limited in vivo.

Project description:BACKGROUND:The ferric uptake regulator (Fur) is a transcription factor and the main regulator of iron acquisition in prokaryotes. When bound to ferric iron, Fur recognizes its DNA binding site and generally executes its function by repressing transcription of its target genes. Due to its importance in virulence, the Fur regulon is well studied for several model bacteria. In our previous work, we used computational predictions and microarray to gain insights into Fur-regulation in Aliivibrio salmonicida, and have identified a number of genes and operons that appear to be under direct control of Fur. To provide a more accurate and deeper global understanding of the biological role of Fur we have now generated an A. salmonicida fur knock-out strain and used RNA-sequencing to compare gene expression between the wild-type and fur null mutant strains. RESULTS:An A. salmonicida fur null mutant strain was constructed. Biological assays demonstrate that deletion of fur results in loss of fitness, with reduced growth rates, and reduced abilities to withstand low-iron conditions, and oxidative stress. When comparing expression levels in the wild-type and the fur null mutant we retrieved 296 differentially expressed genes distributed among 18 of 21 functional classes of genes. A gene cluster encoding biosynthesis of the siderophore bisucaberin represented the highest up-regulated genes in the fur null mutant. Other highly up-regulated genes all encode proteins important for iron acquisition. Potential targets for the RyhB sRNA was predicted from the list of down-regulated genes, and significant complementarities were found between RyhB and mRNAs of the fur, sodB, cysN and VSAL_I0422 genes. Other sRNAs with potential functions in iron homeostasis were identified. CONCLUSION:The present work provides by far the most comprehensive and deepest understanding of the Fur regulon in A. salmonicida to date. Our data also contribute to a better understanding of how Fur plays a key role in iron homeostasis in bacteria in general, and help to show how Fur orchestrates iron uptake when iron levels are extremely low.

Dataset Information

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling.

Publications

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling.

Similar Datasets

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