Karger Publishers, Microbial Physiology, 2(18), p. 102-108, 2010
DOI: 10.1159/000287989
Full text: Unavailable
The <i>cycP </i>gene encoding a periplasmic cytochrome <i>c</i>′ from the denitrifying β-proteobacterium <i>Achromobacter xylosoxidans</i> was characterized. The genes flanking <i>cycP</i> encode components of a mobile genetic element characteristic of the β-proteobacteria, suggesting that <i>cycP</i> has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the <i>cycP</i> gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of <i>cycP</i> appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome <i>c</i>′ in <i>Escherichia coli,</i> which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20–30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from <i>A. xylosoxidans</i>. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem.