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United States Department of Agriculture

Agricultural Research Service

Title: Cloning, Expression, and Characteristics of the Nadh Oxidase Gene (Nox) of Brachyspira (Serpulina) Hyodysenteriae

Authors
item STANTON, THADDEUS
item Sellwood, Richard - INST ANIM HEALTH, UK

Submitted to: Anaerobe
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 8, 1999
Publication Date: N/A

Interpretive Summary: Brachyspira (Serpulina) hyodysenteriae, a corkscrew-shaped bacterium, causes swine dysentery. This worldwide disease is estimated to cost the U.S. pork industry $100 million per year. There are no vaccines. Standard methods for diagnosis are based on culturing the bacterium. These methods are slow and sometimes imprecise. We do not know how B. hyodysenteriae cells are able to damage the swine intestine, producing the severe bloody diarrhea typical of swine dysentery. B. hyodysenteriae cells contain NOX, a chemical thought to protect B. hyodysenteriae cells when they are in the pig's intestine. Our studies have found the gene for NOX and now we can determine whether B. hyodysenteriae needs the gene to damage the swine intestine. We have also found the gene for NOX to be similar in different B. hyodysenteriae strains. This finding holds promise that rapid and reliable tests can be developed for the diagnosis of swine dysentery.

Technical Abstract: Brachyspira (Serpulina) hyodysenteriae cells consume oxygen during growth beneath a 1% O2:99% N2 atmosphere. A major mechanism of O2 metabolism by this anaerobic spirochete is the enzyme NADH oxidase (EC 1.6.99.3). In these studies, the NADH oxidase gene (nox) of B. hyodysenteriae strain B204 was cloned and sequenced. By direct cloning of a HindIII-digested DNA fragment hybridizing 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 transformed with this recombinant plasmid expressed NADH oxidase activity (1.1 umoles 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 (47%) 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. The 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.

Last Modified: 8/27/2014
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