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Rhizobium sigma vegetative-dependent promoters are different from those of enteric bacteria and have never been characterized before. We report here the biochemical characterization of the ORF1-ntrBC promoter of Rhizobium etli. The minimal promoter region was located by means of a transcriptional fusion and further characterized by in vitro transcription and gel retardation experiments. Oligonucleotides used as DNA competitors in runoff transcription experiments allowed the precise localisation of the promoter region. Protein extracts from an ntrC+, but not from an ntrC- strain, inhibited in vitro transcription. The NtrC protein was found to bind specifically to the promoter, where an NtrC binding site overlapping the transcription initiation site, is present.

The DNA-binding activity of the NtrC protein can be demonstrated in gel retardation assays with concentrated protein extracts of Rhizobium etli. Using extracts from either the wild type or a ntrC mutant strain and an antiserum raised against the NtrC protein, we demonstrate specific binding of NtrC to the upstream regulatory region of the glnII gene, where two putative NtrC-binding sites are present. KNO3-grown bacteria contain less NtrC protein and more NtrC-binding activity than NH4Cl-grown bacteria, thus showing that with this protocol it is possible to detect changes in NtrC-binding activity. The advantages of this assay system in comparison with that using pure proteins is discussed.

Permeabilization with cetyl trimethyl ammonium bromide was used to study the post-translational modification of the PII protein in Rhizobium leguminosarum. Upon incubation with radioactive UTP a single band was obtained after SDS-PAGE and autoradiography. RNase resistance and snake venom phosphodiesterase sensitivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein. Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/alpha-ketoglutarate ratio.