Mapping of Rhizobium japonicum nifB-,fixBC-, and fixA-like genes and identification of the fixA promoter

Abstract

New DNA regions likely to be involved in symbiotic nitrogen fixation were identified and mapped, by interspecies hybridization, on cloned DNA from the slowgrowing Rhizobium japonicum strain 110. NifB-and fixBC-like genes were located near the nifH and nifDK operons encoding the nitrogenase polypeptides, whereas a fixA-like gene is not linked to this cluster. NifB was detected by interspecies hybridization with a Klebsiella pneumoniae nifA/nifB probe, and its identity was confirmed due to its homology with the Rhizobium leguminosarum nifB gene. NifB of R. japonicum was located between nifDK and nifH at a distance of 11 kb downstream from the 3′ end of nifK. Using the Rhizobium meliloti fixABC operon as heterologous probe it was found that, in R. japonicum, fixA is distantly separated from fixBC. FixB and fixC are probably contained together in one operon (fixBC). The approximate start of fixB was located 2.8 kb downstream of the 3′ end of nifH. All genes of the nifDK-nifB-nifH-fixBC cluster are transcribed in the same direction. The DNA region harboring fixA was identified in a cosmid clone bank. The promoter and transcription start site of fixA were sequenced and mapped by a nuclease S1 protection experiment. The promoter sequence (5′-ATGGTAC-5bp-TTGCT-3′) is very similar to the nif-type consensus promoter sequence. Hence, the expression of R. japonicum nif and fix genes may be regulated coordinately.