Francois Richaud

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. I. Identification of a lysR gene encoding an activator of the lysA gene.

The synthesis of diaminopimelate decarboxylase, which catalyzes the decarboxylation of diaminopimelate into lysine, is known to be repressed by lysine and induced by diaminopimelate in Escherichia coli K12. Until now only mutations in lysA, the structural gene for diaminopimelate decarboxylase, have been described that lead to a Lys- phenotype. A set of plasmids carrying adjacent inserts of the lysA region was constructed and employed to transform different Lys- mutants. The complementation pattern observed and the corresponding expression of the lysA gene show that in fact the Lys- phenotype can be obtained by mutations in two different and closely linked loci: one being the lysA structural gene, and the other called lysR. We propose that the lysR gene encodes a positive effector required for the full expression of the lysA gene. The synthesis of a hybrid lysA-lacZ protein constructed in vitro was observed to be decreased dramatically in lysR mutants. Moreover, all the regulatory features were lost, indicating that the LysR activator is necessary for the regulation of lysA expression. The gene order is thyA lysA lysR clockwise around 61 minutes on the chromosome, lysA being transcribed counter-clockwise.

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids.

SummaryAgrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the TL-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the TL-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the TL-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the TL-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the TL-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.