Cloning and characteristics of a gene encoding NADH oxidase, a major mechanism for oxygen metabolism by the anaerobic spirocheta, Brachyspira (Serpulina) hyodysenteriae

Brachyspira (Serpulina) hyodysenteriae cells consume oxygen during growth u nder a 1%O-2:99%N-2 atmosphere. A major mechanism of O-2 metabolism by this anaerobic spirochete is the enzyme NADH oxidase (EC 1.6.99.3). In these in vestigations, the NADH oxidase gene (nox) of B, hyodysenteriae strain B204 was cloned,expressed in Escherichia coli, and sequenced. By direct cloning of a Hind III-digested DNA fragment which hybridized with a nor DNA probe a nd by amplification of B204 DNA through the use of inverse PCR techniques, overlapping portions of the nor gene were identified and sequenced. The nor gene and flanking chromosome regions (1.7 kb total) were then amplified an d cloned into plasmid pCRII. Lysates of E. coli cells transformed with this recombinant plasmid expressed NADH oxidase activity (1.1 mu mol NADH oxidi zed/min/mg protein) and contained a protein reacting with swine antiserum r aised against purified B. hyodysenteriae NADH oxidase. The nor ORF (1.3 kb) encodes a protein with a predicted molecular mass of 50 158 kDa. The B, hy odysenteriae NADH oxidase shares significant (46%) amino acid sequence iden tity and common functional domains with the NADH oxidases of Enterococcus f aecalis and Streptococcus mutans, suggesting a common evolutionary origin f or these proteins. Cloning of the B. hyodysenteriae nor gene is an importan t step towards the goal of generating B. hyodysenteriae mutant strains lack ing NADH oxidase and for investigating the significance of NADH oxidase in the physiology and pathogenesis of this anaerobic spirochete. (C) 1999 Academic Press.