Chromosomal location of the Bacillus subtilis aspartokinase II gene and nucleotide sequence of the adjacent genes homologous to uvrC and trx of Escherichia coli

The aspartokinase II (ask) operon of Bacillus subtilis consists of two in-phase overlapping genes that encode the two subunits of the lysine-sensitive isoenzyme of aspartokinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4). Transduction mapping of the ask operon, inactivated by recombinational insertion of a cat marker, indicates a chromosomal location (about 253 degrees) between leuA and aroG. ask is thus remote from aecB, eliminating aecB as a possible locus for the structural gene of aspartokinase II, but close to aecA and uvrB. The nucleotide sequence of a 2 kb DNA fragment just upstream of the ask operon was determined and found to contain two open reading frames. The deduced amino acid sequence of the distal reading frame exhibits extensive homology with Escherichia coli thioredoxin and that of the proximal one, which overlaps with the ask promoter, is homologous to the deduced product of the E. coli uvrC gene. Insertional mutagenesis of the proximal open reading frame led to a mitomycin-sensitive phenotype, consistent with a role in DNA repair. In conjunction with the data of M. Petricek, L. Rutberg & L. Hederstedt [FEMS Microbiology Letters 61, 85-88] our results define the nucleotide sequence of an 8.8 kb segment of the B. subtilis chromosome near 253 degrees and the following order of genes: trx-uvrB-ask-orfX-sdhC-sdhA-sdhB-orfY++ +-gerE.     

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.     

Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168.

The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.     

Structure of the yeast HOM3 gene which encodes aspartokinase

The yeast HOM3 gene has been cloned molecularly by complementation of a HOM3 mutant. The gene is located about 8 kilobase pairs from HIS1 and is present as a single copy in the yeast genome. Mutations in HOM3 result in a requirement for threonine and methionine (or homoserine) for growth and a lack of detectable aspartokinase activity. The nucleotide sequence of HOM3 predicts an enzyme 414 amino acids long that shows homology to the three Escherichia coli aspartokinases, indicating that it is the structural gene for yeast aspartokinase. An approximately 1800-base pair mRNA is transcribed from the HOM3 gene, initiating at several start sites, 80 and 70 base pairs downstream, respectively, from two TATA boxes. Upstream of the TATA boxes is a single TGACTC sequence. This sequence has been shown to be essential for regulation of several genes that encode amino acid biosynthetic enzymes by the general control system. However, no increase in aspartokinase mRNA is observed under general control derepressing conditions.     

Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli

The gene coding for the subunits of aspartokinase II from Bacillus subtilis has been identified in a B. subtilis DNA library and cloned in a bacterial plasmid (Bondaryk, R. P., and Paulus, H. (1984) J. Biol. Chem. 259, 585-591). The introduction of a plasmid carrying the aspartokinase II gene into an auxotrophic Escherichia coli strain lacking all three aspartokinases restored its ability to grow in the absence of L-lysine, L-threonine, and L-methionine. The B. subtilis aspartokinase gene could thus be functionally expressed in E. coli and substitute for the E. coli aspartokinases. Measurement of aspartokinase levels in extracts of aspartokinaseless E. coli transformed with the B. subtilis aspartokinase II gene revealed an enzyme level comparable to that in a genetically derepressed B. subtilis strain. In spite of the high level of aspartokinase, the growth of the transformed E. coli strain was severely inhibited by the addition of L-lysine but could be restored by also adding L-homoserine. This apparently paradoxical sensitivity to lysine was due to the allosteric inhibition of B. subtilis aspartokinase II by that amino acid, a property which was also observed in extracts of the transformed E. coli strain. The synthesis and degradation of the aspartokinase II subunits were measured by labeling experiments in E. coli transformed with the B. subtilis aspartokinase II gene. In contrast to exponentially growing cells of B. subtilis which contained equimolar amounts of the aspartokinase alpha and beta subunits, the transformed E. coli strain contained a 3-fold molar excess of beta subunit. Pulse-chase experiments showed that the disproportionate level of beta subunit was not due to more rapid turnover of alpha subunit, both subunits being quite stable, but presumably to a more rapid rate of synthesis. After the addition of rifampicin, the synthesis of alpha subunit declined much more rapidly than that of beta subunit, indicating that the two subunits were translated independently from mRNA species that differ in functional stability. In conjunction with the results described in the preceding paper which demonstrated that the aspartokinase subunits are encoded by a single DNA sequence, these observations imply that the alpha and beta subunits of B. subtilis aspartokinase II are the products of in-phase overlapping genes.     

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.     

Nucleotide sequence of lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12. Evolutionary pathway leading to three isofunctional enzymes

The lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12 has been cloned and its nucleotide sequence determined. Analysis of the deduced protein sequence (449 amino acid residues) reveals that the entire sequence of aspartokinase III is homologous to the N-terminal part of the two iso- and bifunctional aspartokinase-homoserine dehydrogenases I and II of E. coli. An evolutionary pathway leading to the three molecular species present in the same organism is proposed, and the possible involvement of a highly conserved region in subunit interactions is discussed.     

Sequence of the Bacillus stearothermophilus gene encoding aspartokinase II

The complete nucleotide sequence of the gene encoding aspartokinase (Ask) II from thermophilic Bacillus stearothermophilus has been determined. Degenerate oligodeoxyribonucleotides primed the amplification of a 932-bp gene. This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction using, as template, self-ligating DNA fragments. The deduced amino-acid sequence is 68.7% identical with the sequence of the Bacillus sp. strain MGA3 Ask II     

Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli

In Escherichia coli, thrA, metLM, and lysC encode aspartokinase isozymes that show feedback inhibition by threonine, methionine, and lysine, respectively. In vitro chemical mutagenesis of the cloned lysC gene was used to identify residues and regions of the polypeptide essential for feedback inhibition by lysine. The isolated lysine-insensitive mutants were demonstrated to have missense mutations in amino acid residues 323-352, and at position 250 of aspartokinase III.     

Aspartokinase III, a new isozyme in Bacillus subtilis 168.

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.     

Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.     

Subunit structure of the methionine-repressible aspartokinase II--homoserine dehydrogenase II from Escherichia coli K12.

The quaternary structure of Escherichia coli K12 aspartokinase II--homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr = 176000. It is composed of two subunits having the same molecular weight and the same charge. The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine-containing peptides and the uniqueness of the N-terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II--homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I--homoserine dehydrogenase I.     

Regulation of lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.     

Immunochemical comparison of a bifunctional enzyme, aspartokinase II-homoserine dehydrogenase II, and its two proteolytic fragments.

We report here a comparison between immunochemical properties of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II of E.coli K12 and of its two isolated proteolytic fragments. Both fragments, one inactive and one endowed with homoserine dehydrogenase activity, react with antibodies raised against the native enzyme. Some of the antibodies elicited against the dehydrogenase fragment can recognize regions of this fragment which are not exposed in the entire enzyme.The immunochemical results are used to discuss a simple model in which this bifunctional enzyme is folded up in two domains. The organization of aspartokinase II-homoserine dehydrogenase II is compared to that of another bifunctional enzyme aspartokinase I-homoserine dehydrogenase I with which it shares some sequence homology.     

Nuclear-magnetic-relaxation studies of the interaction of inhibitor with the threonine-sensitive aspartokinase of Escherichia coli.

The nature of the feedback inhibition of the bifunctional enzyme, aspartokinase I-homoserine dehydrogenase I of Escherichia coli was studied using 13C nuclear magnetic resonance (NMR). Since aspartokinase is activated by Mn(II), the interaction of the inhibitor L-threonine (specifically enriched to 90% 13C in the carboxyl carbon) with the metal-enzyme complex was studied. Spin-lattice (T1) and spin-spin (T2) relaxation times were determined by the partially relaxed Fourier transform method and line-width measurements respectively at 20 MHz. The pronounced broadening of the DL-threonine carboxyl peak in the presence of the Mn(II)-enzyme complex indicates that an L-threonine binding site is close to the metal binding site of the kinase active site. The non-identity of (T1)*M and (T2)*M indicates that conditions of fast exchange prevail. The (T1)*M/(T2)*M ratio was used to estimate a correlation time of 2.0 ns for the dipolar interaction at 25 degrees C. An estimate for the distance between Mn(II) and the threonine carboxyl carbon of 4.4 A (0.44 nm) was obtained. This 13C NMR study has thus located one of the two classes of threonine regulatory sites which exist per subunit; the threonine site identified here is at the aspartokinase active site, adjacent to the catalytic metal site.     

Lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Two aspartokinase (ATP:L-aspartate 4-phosphotrasferase, EC 2.7.2.4) enzyme activities have been identified and partially purified from Bacillus brevis. Aspartokinase I is subject to both inhibition and repression by lysine, and has a molecular weight in the region of 110 000. Aspartokinase II is a lysine-stabilised enzyme, inhibited multivalently by lysine plus theonine and has a molecular weight in the region of 95 000. This attern of aspartokinase activity has not been described previously and is unusual in that one end product (lysine) regulates two isoenzymes catalysing the first reaction of a branced biosynthetic pathway. In the absence of lysine, aspartokinase II changes to a more unstable non-inhibitable enzyme. Both enzymes are stabilised by sulphydryl reducing agents and have similar affinities for ATP, aspartate and lysine. However, there is no evidence for a view that they are products of a common gene. Problem concerned with the regulation of aspartokinase activities in Bacillus species are discussed.