A Rhizobium leguminosarum bv. viciae VF39 gene (gabT) encoding a γ-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized. This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex. Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source. 2-Oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvatedependent activity remains unaffected. This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R. leguminosarum bv. viciae VF39. The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis. Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate. Transcriptional regulation is mediated by GabR in a repressor-like manner. During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone. Nodules induced by gabT mutants, however, are still effective in nitrogen fixation.

In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv. viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development. Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions. As a control, a constitutively expressed gusA gene was included. In the nodules of Nop(nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules. Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutant in gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied. In contrast, in Itn- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions. From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont. No morphologically distinct interzone II-III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31). The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule. This is consistent with an altered oxygen barrier previously reported for these nodules. By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.

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The expression of the Rhizobium meliloti C4-dicarboxylic acid permease gene (dctA) is controlled by the sensor DctB and the transcriptional regulator, DctD. The R. meliloti Dct system has been reconstituted in Escherichia coli. Expression of the dctA promoter is DctBD dependent and is induced in the presence of C4-dicarboxylic acids (dCA). Other carbon sources also influence dctA expression. We demonstrate that the cAMP receptor protein (CRP) has a repressive effect on the dctA promoter. A mutated CRP molecule (CRP-H159L), unable to activate catabolic promoters (but still proficient in DNA binding), gives similar results. This suggests that the CRP-cAMP complex represses the dctA promoter activity by direct interaction with the DNA. Direct binding of the CRP-cAMP complex to the dctA promoter was confirmed in vitro by gel mobility-shift assays. Sequence analysis of the dctA promoter indicates that the most likely binding sites for CRP are the two confirmed DctD-binding sites. It is proposed that the CRP-cAMP complex competes with DctD for occupancy of these sites. Since in the presence of CRP-cAMP complex the uninduced levels of dctA expression are reduced, whereas induced levels are largely unaffected, such competition appears to be an essential regulatory feature of dctA expression.

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric j-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.Rhizobia are gram-negative bacteria that are able to enter symbiotic association with legume plants, leading to the formation of N2-fixing root nodules. This process involves differential gene expression between the host plant and bacteria, but the signals involved in the modulation of gene expression are not well understood. Cyclic AMP (cAMP) might play a role in regulating metabolism of rhizobia.