Identification and characterisation of bacterial multi-copper oxidases and their potential role in iron acquisition

Huston, Wilhelmina May (2004). Identification and characterisation of bacterial multi-copper oxidases and their potential role in iron acquisition PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author Huston, Wilhelmina May
Thesis Title Identification and characterisation of bacterial multi-copper oxidases and their potential role in iron acquisition
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Professor Alastair McEwan
Associate Professor Michael Jennings
Total pages 192
Collection year 2004
Language eng
Subjects L
270301 Bacteriology
780105 Biological sciences
Formatted abstract

The presence and function of multi-copper oxidases in bacteria is an emerging area of research; until recently only a few of these enzymes involved in either copper tolerance or manganese oxidation had been described in bacteria. A comprehensive bacterial genome survey was conducted and demonstrated the presence of putative multi-copper oxidases in a wide diversity of bacteria. The homology and phylogenetic relatedness was examined. The amino acid sequence of the putative multi-copper oxidase from Pseudomonas (Ps.) aeruginosa, MCO, was compared with that of CueO, the multicopper oxidase from Escherichia (E.) coli whose structure has been determined by Xray crystallography, to predict the key copper binding ligands. The native form of the enzyme was purified from Ps. aeruginosa and examined in vitro and the multi-copper oxidase was shown to be a ferroxidase. A mco- mutant was generated in Ps. aeruginosa strain PAK and a complement strain was generated by supply of the mco gene in trans in the plasmid pUCPSK. The examination of the ability of the mco-, complement and wild-type strains to oxidise p-phenylenediamine showed the absence of the activity in the mco- strain confirming that the putative mco encodes for a multi-copper oxidase. The examination of the physiology of the strain showed that the MCO is essential for aerobic growth of the bacteria in the presence of ferrous iron as sole iron source: the complement demonstrated a similar growth to that of wild-type under these conditions. The growth in the presence of ferric iron as sole iron source was similar for all strains. Growth under anaerobic conditions in the presence of ferrous iron was similar for the three strains. The mco- strain had a four-fold reduction when compared to that of the wild-type in the ability to uptake ferrous iron from the media under aerobic conditions. The ability to oxidise manganese was not detected in either the Ps. aeruginosa wildtype or mco- strains. The mco- strain showed no difference in copper tolerance to that witnessed in the wild-type strain. These results indicated that the MCO of Ps. aeruginosa is a ferroxidase with an essential role in aerobic ferrous iron acquisition. A putative homolog, mcoL, from Legionella (L.) pneumophila was examined by amino acid homology and the essential copper binding residues were shown to be present in the sequence. The examination of the amino acid sequence of McoL by topology and cellular location prediction programs indicated the likely location of the enzyme in the cytoplasm associated with the cytoplasmic membrane. mcoL::KnR mutant strains were generated in L. pneumophila strain 130b and the mcoL::KnR strains were complemented with the mcoL gene supplied in trans on the vector pMMB2002. The ability to oxidise the multi-copper oxidase substrate 2,6-dimthoxyphenol was examined in all of the strains and the loss of this ability was observed in the mcoL::KnR strains and restored in the complemented strains. The physiology of the strains was examined and the mcoL::KnR strains were unable to grow aerobically on minimal media in the presence of ferrous iron as sole iron source. The mcoL::KnR strains were able to grow, when supplied with ferric iron as sole iron source in a similar manner to that of the wild-type under the same growth conditions. The mcoL::KnR strains did not demonstrate any impairment in copper tolerance when examined on BYE broth. McoL, the multi-copper oxidase of L. pneumophila, has an essential fimction for the aerobic growth of the organism on minimal media in the presence of ferrous iron as sole iron source. The identification and characterisation of a novel bacterial ferrous iron uptake system mediated by a multi-copper oxidase in Ps. aeruginosa and the description of the important function of McoL the multi-copper oxidase of L. pneumophila in aerobic growth in the presence of ferrous iron is presented in this thesis.

Keyword Copper enzymes
Oxidases
Bacterial growth
Iron -- Metabolism
Additional Notes Discontinuous page number from 139-145 in the original thesis.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Fri, 24 Aug 2007, 18:34:25 EST