Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification.
The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.
Harris
Roger L
RL
Department of Molecular Microbiology, John Innes Centre, Norwich, UK.
Eady
Robert R
RR
Hasnain
S Samar
SS
Sawers
R Gary
RG
D000421:Alcaligenes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001400:Azurin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism / Q000494:pharmacology; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D019897:Periplasm / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009566:Nitrates; D009573:Nitrites; D011994:Recombinant Proteins; D001400:Azurin; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing