CONSTRUCTION OF A BIDIRECTIONAL PROMOTER PROBE VECTOR AND ITS USE IN ANALYZING NOD GENE-EXPRESSION IN RHIZOBIUM-LOTI

A broad-host-range bidirectional promoter reporter vector, pSPV4, has been constructed to analyse the activity of cloned divergent regulatory regions. Plasmid pSPV4 contains a pair of divergent promoterless reporter genes (lacZ and gusA) that are bisected by an extensive multiple cloning site. Each reporter gene is preceded by the portable ribosome-binding site of pMP220 [Spaink et al., Plant Mol. Biol. 9 (1987) 27-39], thus ensuring that ribosomal binding should be equally efficient at initiating translation in both directions. The transcriptional fusion vector pSPV4 has a distinct advantage over unidirectional promoter probe vectors in that it can determine the activity of a cloned bidirectional regulatory region simultaneously in both directions using the same set of cells. The relative activity of each of the divergent promoters, and hence the reporter genes, is therefore not a function of the particular growth state of the culture. To demonstrate the utility of pSPV4, the promoter activity of two Rhizobium loti nod regulatory regions were examined. Although the nod gene organisation in this species is unusual, the promoter activity of these two divergent nod regulatory regions is consistent with the conventional mode of nod expression (constitutive towards the regulatory nodD gene and inducible in the divergent nod gene direction). The construction of the bidirectional reporter vector, pSPV4, involved two intermediary plasmids, pSPV1 and pSPV2. Both of these constructs will also be useful for other researchers, especially given the trend towards the utilisation of gusA as a reporter gene.