Analysis and Molecular Characterization of an Unusual Copper Inducible Homeostasis Mechanism in Pseudomonas putida KT2440

Persistent Link:
http://hdl.handle.net/10150/194391
Title:
Analysis and Molecular Characterization of an Unusual Copper Inducible Homeostasis Mechanism in Pseudomonas putida KT2440
Author:
Quaranta, Davide
Issue Date:
2009
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.
Type:
text; Electronic Dissertation
Keywords:
azurin; copper homeostasis; copper sensing; Pseudomonas; queuosine; two component system
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Soil, Water & Environmental Science; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Rensing, Christopher
Committee Chair:
Rensing, Christopher

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleAnalysis and Molecular Characterization of an Unusual Copper Inducible Homeostasis Mechanism in Pseudomonas putida KT2440en_US
dc.creatorQuaranta, Davideen_US
dc.contributor.authorQuaranta, Davideen_US
dc.date.issued2009en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectazurinen_US
dc.subjectcopper homeostasisen_US
dc.subjectcopper sensingen_US
dc.subjectPseudomonasen_US
dc.subjectqueuosineen_US
dc.subjecttwo component systemen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineSoil, Water & Environmental Scienceen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorRensing, Christopheren_US
dc.contributor.chairRensing, Christopheren_US
dc.contributor.committeememberMaier, Raina M.en_US
dc.contributor.committeememberPierson III, Leland Sen_US
dc.contributor.committeememberMcEvoy, Megan M.en_US
dc.identifier.proquest10329en_US
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