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, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.