Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. III. Nucleotide sequence and regulation of the lysR gene.

The complete nucleotide sequence of the lysR gene, which encodes the activatory protein required for lysA expression, has been determined. Bal31 deletions and translational fusions were used to localize the promoter region and the initiator ATG of the lysR gene which encodes a 311 amino acid polypeptide. Both lysA and lysR coding sequences were found to be divergent and separated by a very short intergenic region consisting of 121 base-pairs between the postulated ATGs of the two proteins. Transfer of the whole lysR gene on a plasmid carrying a lysR-lacZ fusion shows that lysR expression is autoregulated by a factor of 7. The same binding site (73 base-pairs fragment) could be involved in both effects of the LysR product, acting simultaneously as an operator for lysR expression and an initiator for lysA expression. The genetic organization of the whole region (4127 base-pairs) is given. A strikingly symmetrical pattern is observed with the four tightly packed galR, lysA, lysR and orfX (an unidentified open reading frame) genes, in a very unusual arrangement of both divergent and convergent overlapping transcription units.

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. II. Nucleotide sequence of the lysA gene and its regulatory region.

The complete nucleotide sequence of the lysA gene and its regulatory region was determined. At the 3' end of the lysA gene an open reading frame was revealed in the opposite direction and was identified as the galR coding region. Only six base-pairs are present between the two translational stops and thus both transcription units are overlapping in vivo. Translational gene fusions constructed in vitro with the beta-galactosidase gene were used to identify the lysA initiating ATG. The sequence encodes a 420 amino acid long peptide for a predicted molecular weight of 46,099. No attenuation-like sequence was found at the beginning of the lysA gene. A target of the LysR activator protein was localized on a 73 base-pair fragment found 48 base-pairs upstream from the lysA coding region. The presence of this DNA sequence on a multicopy plasmid led to a net decrease of lysA expression, indicating limiting amounts of active LysR protein in the cytoplasm.

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.

Isolation and identification of mutants constitutive for aspartokinase III synthesis in Escherichia coli K 12.

We devised a procedure in order to isolate, in Escherichia coli, constitutive mutants for aspartokinase III synthesis, the first enzyme of the lysine regulon. It consists of the introduction of a limiting step in lysine biosynthesis, by the use of the partial suppression of a nonsense mutation. For the first time we could isolate many constitutive mutants. Their characteristics (cotransduction with the lysC structural gene; no effect on the synthesis of other enzymes of the regulon; cis-dominance) lead to classify these mutations as operator-type. The fact that no repressor mutations could be isolated is discussed.

Evolutionary comparisons of three enzymes of the threonine biosynthetic pathway among several microbial species.

As an approach in the study of the evolution of threonine biosynthetic pathways throughout various organisms, the sequences of three enzymes, namely homoserine dehydrogenase, homoserine kinase and threonine synthase, originating from six organisms, namely Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Brevibacterium lactofermentum, Pseudomonas aeruginosa and Saccharomyces cerevisiae, were compared. As a general trend all three enzymatic activities were carried out by proteins sharing sequence relatedness (except for the homoserine kinase of P aeruginosa). Unexpectedly however, for each step one or two enzymes stood out of the main stream: i) for homoserine dehydrogenase, the yeast protein is atypically similar to the E coli enzyme; ii) for homoserine kinase, the P aeruginosa protein shares no similarity with any other species; and iii) for threonine synthase, the B subtilis protein is far distant from the enzymes of other species. Hence in contrast to other biosynthetic pathways such as the tryptophan one, the threonine pathway seems not to have evolved as a whole throughout different organisms but rather each step seems to have been subjected to multiple constraints including substrate-mediated ones and host-specific ones.

Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12.

A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12. With this strain the Kmapp for lysine is 25 fold higher than with the parental strain. The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H). Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase. Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon.

The leader sequence of the Escherichia coli lysC gene is involved in the regulation of LysC synthesis.

In Escherichia coli and Bacillus subtilis, long leader sequences are found upstream of the lysC coding sequences which encode lysine-sensitive aspartokinase. Highly conserved regions exist between these sequences. Mutations leading to constitutive expression of the E. coli lysC gene have been localised within these conserved regions, indicating that they participate in the lysine-mediated repression mechanism of lysC expression.

Multivalent repression of aspartic semialdehyde dehydrogenase in Escherichia coli K-12.

Mutants of Escherichia coli in which the lysine-sensitive aspartokinase is feedback-resistant are described. In these strains, as well as in the wild type, aspartic semialdehyde dehydrogenase is subject to multivalent repression by lysine, threonine, and methionine. When these amino acids were added to a culture in minimal medium, the differential rate of synthesis of the enzyme dropped to zero and remained there for about one generation.

Lysine-sensitive aspartokinase of Escherichia coli K12. Synergy and autosynergy in an allosteric V system.

The interactions of the lysine-sensitive aspartokinase of E. coli K12 with lysine and leucine, as evidenced in the inhibition and binding curves, are well explained by the equations of an allosteric V model. Mathematical treatments of such a model lead to new linearized plots. These representations are applied to our experimental results and allow the direct determination of some parameters of the model (equilibrium constant L' and leucine dissociation constants). The other parameters are obtained by an optimization method. The theoretical curves drawn according to this model account well for the synergistic inhibition between lysine and leucine and for the role of the two nonequivalent lysine binding sites ("autosynergy").

Nucleotide sequence of the promoter region of the E. coli lysC gene.

The regulatory region of the lysC gene (that encodes the lysine-sensitive aspartokinase of Escherichia coli) has been identified and purified by the use of lysC-lacZ fusions. Its regulatory sequence has been determined. No signals similar to those described in the case of an attenuation mechanism could be found in the long leader sequence existing between the starts of transcription and of translation.

Multiple regulatory signals in the control region of the Escherichia coli carAB operon.

The first reaction in pyrimidine and arginine biosynthesis in Escherichia coli is catalyzed by a single enzyme, carbamoyl-phosphate synthetase (EC 6.3.5.5), the product of the carAB operon. Expression of this operon is cumulatively repressed by arginine and pyrimidines. The nucleotide sequence of the carAB control region was determined and transcriptional starts were localized. Two adjacent promoters, 70 base pairs apart, appear to be used in vivo, the downstream one overlapping a typical arginine operator. The absence of any attenuation-like sequence excludes such a mechanism for pyrimidine-mediated repression. Various fragments of the carA promoter-proximal region were fused in vitro with the lacZ gene. Results obtained with these fusions indicate that (i) translation of the carA gene can be initiated in vivo without an AUG codon but very likely with an UUG or an AUU codon; (ii) the carAB downstream promoter is repressed by arginine; and (iii) the carAB upstream promoter is repressed by pyrimidines and subject to stringent control. When carried by a multicopy plasmid the carAB control region escapes repression by arginine and pyrimidines. The existence of a pyrimidine repressor, present in limiting amounts in the cell, is therefore postulated.

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway.

A library of Desulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidly lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridizations, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

In Escherichia coli, the first enzyme of the diaminopimelate and lysine pathway is dihydrodipicolinate synthetase, which is feedback-inhibited by lysine and encoded by the dapA gene. The location of the dapA gene on the bacterial chromosome has been determined accurately with respect to the neighboring purC and dapE genes. The complete nucleotide sequence and the transcriptional start of the dapA gene were determined. The results show that dapA consists of a single cistron encoding a 292-amino acid polypeptide of 31,372 daltons.

Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.

The asd gene of escherichia coli encodes aspartic semialdehyde dehydrogenase, an enzyme involved in lysine, threonine, and methionine biosynthesis; its synthesis is controlled by a multivalent repression mechanism. It was cloned in plasmid pBR322 and its complete nucleotide sequence determined. The sequence predicts a polypeptide chain of 367 amino acids, in good agreement with results obtained for the purified protein ( Biellmann et al., 1980a ). Our data indicate a Cys residue instead of a His residue, which was proposed after covalent labeling of the active center of the enzyme; this is more in line with the catalytic site of glyceraldehyde-3-phosphate dehydrogenase, an enzyme which carries out a similar reaction. The nucleotide sequence that precedes the translational start does not display any of the characteristic features of an attenuation signal. Hence the expression of the asd gene is probably not controlled in the same way as other multivalently repressed operons such as ilva and thr.

Effect of mutations affecting lysyl-tRNAlys on the regulation of lysine biosynthesis in Escherichia coli.

When studying mutants affecting lysyl-tRNA synthetase or tRNAlys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This exlcudes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNAlys is a secondary effector involved in the regulation of the synthesis of this enzyme.

[Regulation of lysyl-tRNA synthetase of Escherichia coli K12]. / Régulation de la lysyl-tARN synthétase d'Escherichia coli K 12

In Escherichia coli K 12, addition of lysine to the growth medium does not lead to a repression of the synthesis of the lysyl-tRNA synthetase. On the contrary, by use of a strain bradytrophic for one of the lysine biosynthetic enzymes, a shift from a medium containing lysine to minimal medium leads to an increase in the specific activity for this enzyme. This increase is discrete (1.5 fold) but reproducible. Thus it may be assumed that lysyl-tRNA synthetase belongs to the lysine regulon.