High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe) as well as nitrogen fixation (nif and fix) are carried on pNGR234a, t he 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NG R234. Putative transcription regulators comprise 24 of the predicted 416 op en reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma(54) regulatory sequenc es, which are thought to co-ordinate the expression of nodulation and nitro gen fixation genes respectively. To analyse transcription of all putative O RFs, the nucleotide sequence of pNGR234a was divided into 441 segments desi gned to represent all coding and intergenic regions, Each of these segments was amplified by polymerase chain reactions, transferred to filters and pr obed with radioactively labelled RNA. RNA was extracted from bacterial cult ures grown under various experimental conditions, as well as from bacteroid s of determinate and indeterminate nodules, Generally, genes involved in th e synthesis of Nod factors (e.g, the three hsn loci) were induced rapidly a fter the addition of flavonoids, whereas others thought to act within the p lant (e.g. those encoding the type III secretion system) responded more slo wly. Many insertion (IS) and transposon (Tn)like sequences were expressed s trongly under all conditions tested, while a number of loci other than thos e known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules, Many more diverse transcripts were found in bacteroids of determi nate as opposed to indeterminate nodules.