Cloning and Characteristics of a Gene Encoding NADH Oxidase, a Major Mechanism for Oxygen Metabolism by the Anaerobic Spirochete, Brachyspira (Serpulina) hyodysenteriae

Brachyspira (Serpulina) hyodysenteriae cells consume oxygen during growth under a 1%O2:99%N2atmosphere. A major mechanism of O2metabolism by this anaerobic spirochete is the enzyme NADH oxidase (EC 1.6.99.3). In these investigations, the NADH oxidase gene (nox) of B. hyodysenteriae strain B204 was c...

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Bibliographic Details
Published in:Anaerobe 1999-10, Vol.5 (5), p.539-546
Main Authors: Stanton, Thad B, Sellwood, Richard
Format: Article
Language:English
Subjects:
amino acid sequences
animal pathogenic bacteria
bacterial proteins
Brachyspira hyodysenteriae
dysentery
gene expression regulation
molecular cloning
molecular sequence data
NADH or NADPH oxidoreductases
nucleotide sequences
oxidative stress, swine dysentery, intestinal bacteria, anaerobe
oxygen
sequence analysis
swine diseases
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Summary:Brachyspira (Serpulina) hyodysenteriae cells consume oxygen during growth under a 1%O2:99%N2atmosphere. A major mechanism of O2metabolism by this anaerobic spirochete is the enzyme NADH oxidase (EC 1.6.99.3). In these investigations, the NADH oxidase gene (nox) of B. hyodysenteriae strain B204 was cloned, expressed in Escherichia coli, and sequenced. By direct cloning of aHind III-digested DNA fragment which hybridized with a nox DNA probe and by amplification of B204 DNA through the use of inverse PCR techniques, overlapping portions of the nox gene were identified and sequenced. The nox gene and flanking chromosome regions (1.7 kb total) were then amplified and cloned into plasmid pCRII. Lysates of E. coli cells transformed with this recombinant plasmid expressed NADH oxidase activity (1.1 μmol NADH oxidized/min/mg protein) and contained a protein reacting with swine antiserum raised against purified B. hyodysenteriae NADH oxidase. The nox ORF (1.3 kb) encodes a protein with a predicted molecular mass of 50 158 kDa. The B. hyodysenteriae NADH oxidase shares significant (46%) amino acid sequence identity and common functional domains with the NADH oxidases of Enterococcus faecalis and Streptococcus mutans, suggesting a common evolutionary origin for these proteins. Cloning of the B. hyodysenteriae nox gene is an important step towards the goal of generating B. hyodysenteriae mutant strains lacking NADH oxidase and for investigating the significance of NADH oxidase in the physiology and pathogenesis of this anaerobic spirochete.
ISSN:1075-9964
1095-8274
1095-8274
DOI:10.1006/anae.1999.0196