Non nod Gene Expression in Rhizobia During Exposure to Aromatic Compounds

RAP- PCR (Welsh et al, 1992), applied to uninduced and daidzein - induced cultures of Rhizobium sp. NGR234, identified a number of plasmid and chromosomal borne loci with significantly increased expression in the presence of the isoflavonoid. These included a 254 bp fragment which has 93% homology to a flavonoid-inducible cultivar specificity gene in R. fredii (nolU) and a 310 bp fragment which exhibits high homology to a number of ATP binding proteins from various bacteria. Another RAP-PCR product with increased expression in the presence of daidzein shared 99% homology with the polyphosphate kinase gene from E. coli (ppk) over 302 of its 308 base pair length. Hybridisation of a cosmid library digest of the NGR234 symbiotic plasmid (Perret et al, 1991) with labelled RNA from uninduced or daidzein - induced NGR234 cultures also confirmed that the expression of many genes, in addition to nod genes, was induced by this isoflavone. In Agrobacterium tumefaciens the monocyclic aromatic protocatechuate is metabolised via the protocatechuate branch of the beta - ketoadipate pathway, involving five structural genes (pcaDCHGB) in a single operon under the control of a regulatory gene, pcaQ (Parke, 1996). In view of the fact that other members of the Rhizobiaceae can utilize protocatechuate as a sole carbon source it seems likely that the same mechanism is involved. We have adopted two different approaches to investigate the presence of genes for the metabolism of this compound in Rhizobium sp.NGR234. Degenerate primer PCR was used to target protocatechuate 3, 4 dioxygenase (pcaHG) and this yielded a 291 bp sequence with significant homology to pcaHG from Acinetobacter calcoaceticus. Secondly, a pcaQ DNA probe from Agrobacterium tumefaciens hybridised to a 6kb and a 4kb fragment in a Southern blot of an EcoR1 digest of Rhizobium sp. NGR234 genomic DNA.