px(2), the newly identified gene in Rhizobium leguminosarum, is characterized to enhance its adjacent nodF expression.

nodFEL operon is one of the NodD-dependent inducible nod operons that is clustered on the symbiosis plasmid of Rhizobium leguminosarum biovar viciae. A recent study on the upstream regulatory region of nodFEL operon had identified a new promoter, which was responsible for the transcription of a 0.72 Kb RNA molecule, called px(2), in the opposite direction to nodF. This new promoter was further characterized to overlap with that of nodF and its in vitro transcription was inhibited by another newly identified nod regulator, Px. In this paper we report that the sequence analysis of the px(2) transcript revealed only one open reading frame (ORF(66)), corresponding to a polypeptide of 66 amino acids. Moreover, the increase in px(2) copy numbers enhanced the in vivo inducible expression level of nodF, whereas the frame-shift mutation of ORF(66) eliminated such effect, providing evidence that px(2) is responsible for specifically upregulating nodF expression. This result also raises the big possibility that px(2) encodes this polypeptide. A model for coordinated expression of px(2) and nodF, transcribed divergently from each other, is proposed.