Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum

The structural gene. nirK. for the respiratory Cu-containing nitrite reduct ase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to ot her Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the seque nce upstream from the structural nirK gene revealed the presence of an anae robox located 83 base pairs from the putative translational start codon. Ce lls of strain GRK308, a nitrite reductase-deficient derivative of strain US DA110, were unable to grow when cultured under microaerobic conditions (1% O-2) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level o xygen conditions and the presence of nitrate. Expression of beta -galactosi dase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubatio n of the cells under oxy en-limiting conditions either with or without nitr ate. Complementation of B. japonicum 9043 with the fixK(2) gene restored be ta -galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxyg en-responsive two-component regulatory system FixLJ and on the Fnr/FixK-lik e DNA binding protein FixK(2). (C) 2001 Elsevier Science B.V. All rights re served.