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Copper homeostasis and nitrosative stress in Lactococcus lactis

English title Copper homeostasis and nitrosative stress in Lactococcus lactis
Applicant Solioz Marc
Number 122551
Funding scheme Project funding (Div. I-III)
Research institution Institut für Klinische Pharmakologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.10.2008 - 30.09.2011
Approved amount 439'286.00
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All Disciplines (2)

Discipline
Biochemistry
Experimental Microbiology

Keywords (8)

copper homeostasis; regulation; transport; proteolysis; nitrosative stress; nitrosylation; heavy metals; stress

Lay Summary (English)

Lead
Lay summary
Although copper is an essential trace element, it is toxic when present in excess. To control cytoplasmic copper levels, all living organisms have evolved homeostatic mechanisms. In the bacterial species studied thus far, a key element in the control of cytoplasmic copper are copper-exporting ATPases. One or two genes encoding such enzymes can be found in bacterial genomes. The expression of the copper ATPases is regulated by copper via copper responsive repressors. In additions, copper chaperones involved in the intracellular routing of copper are usually present.To identify additional, novel genes involved in copper homeostasis, we will employ the food-grade organism Lactococcus lactis. It offers a relatively simple genome which has been sequenced and the organism is of great biotechnological importance. We had earlier applied proteomics to L. lactis. This lead to the identification of the CopR regulon: a set of genes and operons under the control of the copper-responsive CopR repressor. The CopR regulon consists of monocistronic genes and polycistronic operons, encompassing 11 to 14 genes. Some of the genes were already shown to be induced by copper. The function of six genes of the CopR regulon has been characterized to some extent. The copRZA operon encodes the CopR repressor of the regulon, a copper chaperone, CopZ, and a copper ATPase, CopA, which exports copper from the cytoplasm. The lctO gene encodes a lactate oxidase which can serve in the elimination of molecular oxygen under copper stress. The ytjD gene finally appears to be involved in the control of protein denitrosylation under copper stress. AimsThe function of several genes of the CopR regulon remains unknown. To elucidate the role of these genes in copper homeostasis or copper stress is the overall goal of the current proposal. The connection between copper stress and nitrosative stress is novel and will receive special attention. We also propose targeted approaches which should lead to the identification of the copper reductase of L. lactis and the copper-induced protease which degrades the CopZ copper chaperone under high copper conditions.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
ICA Workshop 28.10.2011 Munich, Germany
Wümek Medizinalmesse 24.05.2011 Würzburg, Germany
Deutsches Kupferinstitut Workshop 29.03.2011 Düsseldorf, Germany
Seminar at the University of Dresden 03.03.2011 Dresden, Germany
Wissen heilt 26.02.2011 Basel, Switzerland
Hochschulkonferenz Saarbrücken 11.11.2010 Saarbrücken, Deutschland


Knowledge transfer events



Self-organised

Title Date Place
7the International Copper Meeting 10.10.2010 Alghero, Sardinai, Italy

Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) Antimicrobial Copper Private TV recording International 28.10.2011

Awards

Title Year
NF Postdoctoral fellowship 2011
EMBO short term fellowship 2010

Use-inspired outputs


Start-ups

Name Year
CopperPen 2011

Associated projects

Number Title Start Funding scheme
109703 How Bacteria deal with copper 01.10.2005 Project funding (Div. I-III)
117446 Fast, accurate, and sensitive mass spectrometry for basic research in life sciences at the university of bern / mass spectrometer 01.10.2007 R'EQUIP

Abstract

BackgroundAlthough copper is an essential trace element, it is toxic when present in excess. To control cytoplasmic copper levels, all living organisms have evolved homeostatic mechanisms. In the bacterial species studied thus far, a key element in the control of cytoplasmic copper are copper-exporting ATPases. One or two genes encoding such enzymes can be found in bacterial genomes. The expression of the copper ATPases is regulated by copper via copper responsive repressors. In additions, copper chaperones involved in the intracellular routing of copper are usually present.To identify additional, novel genes involved in copper homeostasis, we will employ the food-grade organism Lactococcus lactis. It offers a relatively simple genome which has been sequenced and the organism is of great biotechnological importance. We had earlier applied proteomics to L. lactis. This lead to the identification of the CopR regulon: a set of genes and operons under the control of the copper-responsive CopR repressor. The CopR regulon consists of monocistronic genes and polycistronic operons, encompassing 11 to 14 genes. Some of the genes were already shown to be induced by copper. The function of six genes of the CopR regulon has been characterized to some extent. The copRZA operon encodes the CopR repressor of the regulon, a copper chaperone, CopZ, and a copper ATPase, CopA, which exports copper from the cytoplasm. The lctO gene encodes a lactate oxidase which can serve in the elimination of molecular oxygen under copper stress. The ytjD gene finally appears to be involved in the control of protein denitrosylation under copper stress. AimsThe function of several genes of the CopR regulon remains unknown. To elucidate the role of these genes in copper homeostasis or copper stress is the overall goal of the current proposal. The connection between copper stress and nitrosative stress is novel and will receive special attention. We also propose targeted approaches which should lead to the identification of the copper reductase of L. lactis and the copper-induced protease which degrades the CopZ copper chaperone under high copper conditions. ApproachesCharacterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. The ground work for the characterization of novel copper homeostatic genes involves a number of standard tools: targeted gene knock-out, study of the in vivo effects of gene knock-out, overexpression and purification of novel gene products, and study of the function of purified proteins in vitro. More specific methods will be applied as required.To tackle the connection of copper stress and nitrosative stress, changes in intracellular nitrosoglutathione levels in response to copper will be assessed. In vivo nitrosylated proteins will be identified by mass spectroscopy, either directly from gels or following affinity purification on anti-S-nitrosocysteine antibody columns. The action of the YtjD nitroreductase on in vivo or in vitro nitrosylated proteins will be studied. Identification of the copper reductase will be tackled by random mutagenesis of L. lactis by plasmid integration, followed by molecular characterization of the mutants. Alternatively, putative reductase genes of L. lactis identified by bioinformatics will be inactivated by targeted gene knock-out, followed by phenotypic analysis.SignificanceThe studies outlined in this proposal should reveal new mechanisms of how bacteria deal with copper stress. Importantly, the connection of copper stress and nitrosative stress opens new avenues in the metal stress area. Given the evolutionary conservation of some components of the copper homeostatic machinery, new components and mechanisms of copper homeostasis revealed in L. lactis may be of broader significance.
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