Bert Boesten

Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport.

The genes controlling the transport of C4-dicarboxylic acids from Rhizobium meliloti have been cloned and analysed. The nucleotide sequence of the control region of the structural dctA and the regulatory dctBD genes has been determined. Comparison with the Rhizobium leguminosarum dct genes revealed a high degree of homology. Gene fusions to the enteric lacZY reporter gene were constructed and the expression of the dctA and dctBD genes studied under various physiological conditions. In free-living cells, the regulatory dctBD genes are absolutely required for the expression of the dctA gene. In the root nodule environment, a dctA::lacZY gene fusion was found to be expressed in an R. meliloti strain mutated in both the dctB and dctD genes, but not in a strain mutated in the dctB gene alone. The presence of the conserved upstream NifA-binding sites on the dctA promoter sequence, coupled with the fact that the dctA::lacZY gene fusion is not expressed in root nodules formed by a nifA mutant strain of R. meliloti, supports the suggestion that NifA may be involved in the symbiotic expression of dctA in the absence of the regulatory dctBD genes. Under micro-aerobic conditions, however, NifA induction alone is not sufficient for expression of the dctA promoter, even though the NifA-dependent nifHDK promoter is highly expressed under these conditions.

Effect of Mutations in Pisum sativum L. Genes Blocking Different Stages of Nodule Development on the Expression of Late Symbiotic Genes in Rhizobium leguminosarum bv. viciae

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.

The Escherichia coli cAMP receptor protein (CRP) represses the Rhizobium meliloti dctA promoter in a cAMP‐dependent fashion

The expression of the Rhizobium meliloti C4‐dicarboxytic 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‐dicarboxyrtc 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 ONA 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.

DctBD-Dependent and -Independent Expression of the Sinorhizobium (Rhizobium) meliloti C4-Dicarboxylate Transport Gene (dctA) During Symbiosis

The Sinorhizobium meliloti C4-dicarboxylate transport gene (dctA) is essential for symbiotic nitrogen fixation. Under free-living conditions, the expression of dctA is fully dependent on the cognate regulatory genes dctBD. However, during symbiosis with the Medicago sativa host plant, the dctA gene is efficiently expressed even in the absence of the dctBD genes. The spatial expression of the dctA gene has been monitored in situ in mature nitrogen-fixing nodules formed by wild-type and dctD mutant strains. In nodules induced by a wild-type strain, expression was observed in both the infection zone and the nitrogen-fixing zone of the nodule. DctD-independent expression of dctA was observed with a previously described dctA∷lacZ fusion (pCU700) and was found to be confined to the fixation zone (zone III) of mature nodules. Therefore, the operation of the alternative system of symbiotic dctA activation (ASA) is concomitant with the onset of nitrogen fixation, which could be consistent with an increased need fo...

Nucleotide sequence of a tRNAleuCAG gene from Rhizobium meliloti

The nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti has been determined. Its organization includes a short transcript with a promoter-like structure and two Rho-independent terminators about 10 bp downstream from the cloverleaf structure. The putative promoter shows good homology with the -45 and -35 region of the consensus (sigma 70) promoter sequence of Escherichia coli, but less with the -10 region. A discriminator-like sequence is present between the start of the transcript and the -10 region. The gene shows extensive homology to tRNA(leu)CAG genes from Gram-positive and Gram-negative bacteria. However, significant differences are encountered in the variable loop region. This is the first report of a tRNA sequence from R. meliloti.