Characterization of copper mediated transcriptional responses in Bacillus subtilis and Identification of copper and iron connection in Bacillus subtilis.

Copper is an essential cofactor for many enzymes and over a threshold, it is toxic for all organisms. The redox ability of copper to cycle between two oxidation states, Cu(I) and Cu(II) makes it crucial for the structure and function numerous enzymes. Uncontrolled accumulation of copper inside the c...

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1. Verfasser: Chillappagari, Shashipavan
Beteiligte: Bölker, M. (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2009
Biologie
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Zusammenfassung:Copper is an essential cofactor for many enzymes and over a threshold, it is toxic for all organisms. The redox ability of copper to cycle between two oxidation states, Cu(I) and Cu(II) makes it crucial for the structure and function numerous enzymes. Uncontrolled accumulation of copper inside the cell leads to the generation of reactive oxygen species (ROS) through fenton reaction leading to oxidative stress and finally damages DNA and proteins. In this work, the gram positive bacterium Bacillus subtilis was used to understand the mechanisms underlying copper homeostasis. Especially, the recently identified copper efflux transcriptional regulator CsoR was mainly in focus. Deletion mutant of ΔcsoR shows a growth defective phenotype in copper excess conditions and exhibited a slight better growth phenotype under copper limiting conditions. Microarray studies initiated with ΔcsoR under copper limiting conditions revealed the ycnJ gene to encode a protein that plays an important role in copper metabolism, as it shows a significant 8-fold upregulation under copper limiting conditions, and its disruption exhibits a growth defective phenotype under copper limiting as well as copper excess conditions. Native gel shift experiments with the recombinant Nterminal cytosolic domain of the YcnJ membrane protein (135 residues) disclose its strong affinity to Cu(II) atoms in vitro. Inspection of the upstream sequence of ycnJ revealed the ycnK gene to encode a putative transcriptional regulator, whose deletion showed a constitutive expression of ycnJ. Further studies supported a predominant role for the YcnJ protein not only as a copper importer under copper limiting conditions, but also possibly as a copper resistance determinant under copper excess conditions. Interestingly, the correlation between copper dependent iron aquisition was also elucidiated. Microarray approach to identify the copper responsive genes using ΔcsoR mutant exhibited an alternate regulation of iron responsive genes in the presence or absence of copper. Significant downregulation of the following iron responsive genes was observed: 1. dhbABCEF which is involved in bacillibactin production, 2. feuABC which are responsible for bacillibactin uptake and 3. besA and btr, which are involved in sensing iron bound bacilibactin, hydrolysis, cleavage and subsequent release of iron from bacillibactin. Transcription profiling data, quantification of bacillibactin amounts and estimation of total cellular iron and copper concentrations in wildtype and the ΔcsoR mutant in response to different copper amounts demonstrated an indirect relation between copper and iron. Further, ΔcsoR mutant exhibited differential regulation of oxygen sensing, cytochromes and anaerobic genes in response to excess copper in the growth media suggesting a probable role for CsoR and its significance in energy generation, cellular respiration, oxygen transport, and iron acquisition under different oxygen availability conditions imposed by the presence of copper.
DOI:https://doi.org/10.17192/z2010.0072