Dihydrodipicolinate Synthase Research Articles

The Crystal Structure of Three Site-directed Mutants of Escherichia coli Dihydrodipicolinate Synthase: Further Evidence for a Catalytic Triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of ( S)-aspartate-β-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5 Å, revealing a tetrameric molecule comprised of four identical (β/α) 8-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35 Å resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.

Dihydrodipicolinate synthase is not inhibited by its substrate, (S)-aspartate beta-semialdehyde.

DHDPS (dihydrodipicolinate synthase; EC 4.2.1.52) is the enzyme that catalyses the first unique step of lysine biosynthesis in plants and micro-organisms. As such, it has attracted much attention as a target for herbicide and anti-microbial action. DHDPS has two substrates: pyruvate and ( S )-aspartate beta-semialdehyde [( S )-ASA]. There are various literature reports that suggest that high levels of ( S )-ASA inhibit the enzyme [Karsten (1997) Biochemistry 36, 1730-1739; Stahly (1969) Biochim. Biophys. Acta 191, 439-451], whereas others have not observed this phenomenon. We have resolved this long-running literature debate and shown unequivocally that this difference in reported behaviour can be attributed to differences in the preparation of ( S )-ASA used by each researcher. DHDPS is not inhibited by its substrate; rather, the inhibition is due to an, as yet, unidentified inhibitor in preparations of the substrate generated by ozonolysis. Furthermore, we demonstrate that ( R )-ASA is neither an inhibitor nor a substrate of DHDPS from Escherichia coli.

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus.

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.

MosA, a Protein Implicated in Rhizopine Biosynthesis in Sinorhizobium meliloti L5-30, is a Dihydrodipicolinate Synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3- O-methyl- scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure–function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate β-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by l-lysine are consistent with the enzyme being a DHDPS. E. coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.

Mimicking natural evolution in vitro: an N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity.

N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (betaalpha)(8)-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself. In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant k(cat)K(M) for the new substrate l-aspartate-beta-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 A resolution in complex with the inhibitor beta-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps, nature has exploited the catalytic promiscuity of many enzymes to evolve novel enzymes or biological pathways during the course of evolution.

Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds.

To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained approximately 12-fold or approximately 5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic approximately 80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to approximately 38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.

Coexpression of the soybean vegetative storage protein beta subunit (S-VSPbeta) either with the bacterial feedback-insensitive dihydrodipicolinate synthase or with S-VSPalpha stabilizes the S-VSPbeta transgene protein and enhances lysine production in transgenic tobacco plants.

Soybean vegetative storage proteins (S-VSPs) are lysine-rich leaf proteins, originally found to accumulate to high levels in depodded soybean plants. In the present study, we overexpressed S-VSPbeta, the ruminant stable subunit of the S-VSP genes, in transgenic tobacco plants. The S-VSPbeta protein accumulated in all organs studied, but its level declined drastically with leaf age. This instability of S-VSPbeta could be overcome either by elevating free lysine levels or by coexpressing S-VSPbeta with S-VSPbeta. High levels of rumen-stable, lysine-rich proteins is expected to improve absorption of lysine by ruminants. Furthermore, the expression of S-VSPs in heterologous plants led to a significant increase in total soluble lysine, suggesting that these proteins may also permit better assimilation of lysine by humans and monogastric animals.

Biotechnological manufacture of lysine.

L-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years. Nowadays production exceeds 600,000 tons per year. Based on conventionally bred strains, further improvement of lysine productivity has been achieved by genetic engineering. Pyruvate carboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase and the specific lysine exporter were shown to be key enzymes for lysine production and were characterized in detail. Their combined engineering led to a striking increase in lysine formation. Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinated flux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cycling between C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle. Process economics have been optimized by developing repeated fed-batch techniques and technical continuous fermentations. In addition, on-line metabolic pathway analysis or flow cytometry may help to improve the fermentation performance. Finally, the availability of the Corynebacterium glutamicum genome sequence has a major impact on the improvement of the biotechnological manufacture of lysine. In this context, all genes of the carbon flow from sugar uptake to lysine secretion have been identified and are accessible to manipulation. The whole sequence information gives access to post genome technologies such as transcriptome analysis, investigation of the proteome and the active metabolic network. These multi-parallel working technologies will accelerate the generation of knowledge. For the first time there is a chance of understanding the overall picture of the physiological state of lysine overproduction in a technical environment.

Analysis of Dihydrodipicolinate Synthase Promoter Activity in Arabidopsis thaliana by In-Planta Transformation

The present study was carried out to analyze the dihydrodipicolinate synthase (dhdps) gene promoter activity by tracing the GUS expression in tissues and in organs of Arabidopsis thaliana by in planta transformation. The Agrobacterium construct pBI101 used in the studies consists of the reporter gene gus under the control of Arabidopsis thaliana dhdps promoter with 3’ nos controlling sequences and nptll gene under the control of nos promoter and nos terminator. GUS expression in transformed Arabidopsis thaliana was found to be cell type-specific and expressed mainly in the fast growing tissues, where the protein synthesis is high. The histochemical analysis results indicate that the GUS expression was mainly observed in root meristem (elongation zone), emerging lateral roots and in the leaf vascular tissues. In reproductive organs, the GUS expression was observed in anthers, pollen grains and young immature embryos. Southern blot analysis results of T2, progeny showed the presence of a single integration locus for both the nptll and dhdps promoter.The segregation analysis results showed that the kanamycin resistance gene has not followed the normal Mendelian inheritance.This might be due to the methylation of the nptll gene in some of the transformants.

Increased Production of Nutriments by Genetically Engineered Crops

Plants are the basis of human nutrition and have been selected and improved to assure this purpose. Nowadays, new technologies such as genetic engineering and genomics approaches allow further improvement of plants. We describe here three examples for which these techniques have been employed. We introduced the first enzyme involved in fructan synthesis, the sucrose sucrose fructosyltransferase (isolated from Jerusalem artichoke), into sugar beet. The transgenic sugar beet showed a dramatic change in the nature of the accumulated sugar, 90% of the sucrose being converted into fructan. The use of transgenic sugar beet for the production and isolation of fructans will result in a more efficient plant production system of fructans and should promote their use in human food. The second example shows how the over-expression of the key enzyme of flavonoid biosynthesis could increase anti-oxidant levels in tomato. Introduction of a highly expressed chalcone isomerase led to a seventyfold increase of the amount of quercetin glucoside, which is a strong anti-oxidant in tomato. We were also able to modify the essential amino acid content of potato in order to increase its nutritional value. The introduction of a feedback insensitive bacterial gene involved in biosynthesis of aspartate family amino acids led to a sixfold increase of the lysine content. Because the use of a bacterial gene could appear to be controversial, we also introduced a mutated form of the plant key enzyme of lysine biosynthesis (dihydrodipicolinate synthase) in potato. This modification led to a 15 times increase of the lysine content of potato. This increase of the essential amino acid lysine influences the nutritional value of potato, which normally has low levels of several essential amino acids. These three examples show how the metabolism of primary constituents of the plant cell such as sugar or amino acids, but also of secondary metabolites such as flavonoids, can be modified by genetic engineering. Producing fructan, a soluble fiber, increasing the level of flavonoids, an antioxidant, in tomato or increasing the level of essential amino acids in potato are all clear examples of plant genetic modifications with possible positive effects on human nutrition.

Metabolic engineering of a complex biochemical pathway: The lysine and threonine biosynthesis as an example

The nutritional quality of crop plants is determined by their content in essential amino acids provided in food for humans or in feed for monogastric animals. Amino acid composition of crop–based diets can be improved via manipulation of the properties of key enzymes of amino acid biosynthetic pathways by mutation and transformation. We focused on the aspartate-derived amino acid pathway producing four essential amino acids: lysine, threonine, isoleucine and methionine. Genes encoding aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) that operate as key genes of the aspartate pathway have been cloned from Arabidopsis. Genetic and molecular studies revealed that at least five different ak genes are represented. Some of them were characterized in terms of gene and promoter structure, developmental expression and regulatory properties. In the case of dhdps, two quite identical genes have been identified and characterized at expression level. Mutated genes encoding a fully feedback-insensitive form of the DHDPS enzyme were obtained from Nicotiana sylvestris and Arabidopsis. Several chimeric constructs harbouring this mutated allele under the control of constitutive or seed-specific promoters were transferred via Agrobacterium or biolistics in various plant species. In all cases, lines with significant increase of free lysine content were obtained in vegetative organs, but the impact of the transgene in seeds is limited due to the presence of an active catabolic enzyme, lysine ketoreductase. These results show that, although dealing with a complex, highly regulated pathway, the overexpression of a single gene encoding a feedback-insensitive form of the key enzyme DHDPS exerts a significant effect on the carbon flux through the aspartate pathway towards lysine production.

Overproduction of L-Lysine from methanol by Methylobacillus glycogenes derivatives carrying a plasmid with a mutated dapA gene.

The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.

Neighboring cysteine residues in human fucosyltransferase VII are engaged in disulfide bridges, forming small loop structures

Among alpha 3-fucosyltransferases (alpha3-FucTs) from most species, four cysteine residues appear to be highly conserved. Two of these cysteines are located at the N-terminus and two at the C-terminus of the catalytic domain. FucT VII possesses two additional cysteines in close proximity to each other located in the middle of the catalytic domain. We identified the disulfide bridges in a recombinant, soluble form of human FucT VII. Potential free cysteines were modified with a biotinylated alkylating reagent, disulfide bonds were reduced and alkylated with iodoacetamide, and the protein was digested with either trypsin or chymotrypsin, before characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry. More than 98% of the amino acid sequence for the truncated enzyme (beginning at amino acid 53) was verified. Mass spectrometry analysis also demonstrated that both potential N-linked sites are occupied. All six cysteines in the FucT VII sequence were shown to be disulfide-linked. The pairing of the cysteines was determined by proteolytic cleavage of nonreduced protein and subsequent analysis by mass spectrometry. The results demonstrated that Cys(68)-Cys(76), Cys(211)-Cys(214), and Cys(318)-Cys(321) are disulfide-linked. We have used this information, together with a method of fold recognition and homology modeling, using the (alpha/beta)(8)-barrel fold of Escherichia coli dihydrodipicolinate synthase as a template to propose a model for FucT VII.

Lysine metabolism in higher plants.

The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine a-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/ dephosphorylation.

Aspartate kinase 2. A candidate gene of a quantitative trait locus influencing free amino acid content in maize endosperm.

The maize (Zea mays) Oh545o2 inbred accumulates an exceptionally high level of free amino acids, especially lysine (Lys), threonine (Thr), methionine, and iso-leucine. In a cross between Oh545o2 and Oh51Ao2, we identified several quantitative trait loci linked with this phenotype. One of these is on the long arm of chromosome 2 and is linked with loci encoding aspartate (Asp) kinase 2 and Asp kinase (AK)-homoserine dehydrogenase (HSDH) 2. To investigate whether these enzymes can contribute to the high levels of Asp family amino acids, we measured their specific activity and feedback inhibition properties, as well as activities of several other key enzymes involved in Lys metabolism. We did not find a significant difference in total activity of dihydrodipicolinate synthase, HSDH, and Lys ketoglutarate reductase between these inbreds, and the feedback inhibition properties of HSDH and dihyrodipicolinate synthase by Lys and/or Thr were similar. The most significant difference we found between Oh545o2 and Oh51Ao2 is feedback inhibition of AK by Lys but not Thr. AK activity in Oh545o2 is less sensitive to Lys inhibition than that in Oh51Ao2, with a Lys I50 twice that of Oh51Ao2. AK activity in Oh545o2 endosperm is also higher than in Oh51Ao2 at 15 d after pollination, but not 20 d after pollination. The results indicate that the Lys-sensitive Asp kinase 2, rather than the Thr-sensitive AK-HSDH2, is the best candidate gene for the quantitative trait locus affecting free amino acid content in Oh545o2.

Molecular genetic dissection and potential manipulation of lysine metabolism in seeds

Summary Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.

Constitutive and seed-specific expression of a maize lysine-feedback-insensitive dihydrodipicolinate synthase gene leads to increased free lysine levels in rice seeds

In order to improve the nutritional value of rice, we prepared transgenic rice plants with a lysine-feedback-insensitive maize dhps gene under the control of CaMV 35S and the rice glutelin GluB-1 promoter for over-expression and seed-specific expression. The transgenic plants were fertile and expressed the dhps gene abundantly or specifically in rice seeds. The transgenic lines (TC lines) containing mutated dhps controlled by CaMV 35S promoter possessed higher mutated DHPS transcript levels and in vitro DHPS activities in seeds than those of TS lines containing the mutated dhps gene driven by a seed-specific promoter, GluB-1. The content of free lysine in immature seeds of both TC and TS lines was higher than that of wild-type plants. The content of free lysine in mature seeds of TC lines was still higher than, but that of TS lines was similar to, that of wild-type plants. From a comparison of DHPS and lysine-ketoglutarate reductase (LKR) expression levels we conclude that the presence of the foreign dhps gene leads to an increase of LKR activity, resulting in enhanced lysine catabolism. However, over-expression of the mutant dhps gene in a constitutive manner overcomes lysine catabolism and sustains a high lysine level in mature rice seeds.

Arabidopsis loss-of-function mutant in the lysine pathway points out complex regulation mechanisms

In plants, the amino acids lysine, threonine, methionine and isoleucine have L-aspartate-β-semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein-bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine-tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T-DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross-pathway regulations are discussed.

Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene.

A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.

Characterization of the Sinorhizobium meliloti genes encoding a functional dihydrodipicolinate synthase (dapA) and dihydrodipicolinate reductase (dapB).

In bacteria, the known pathways for diaminopimelate (DAP) and lysine biosynthesis share two key enzymes, dihydrodipicolinate synthase and dihydrodipicolinate reductase, encoded by the dapA and dapB genes, respectively. In rhizobia, these genes have not yet been genetically characterized. In this work, by sequence analysis, we identified two divergent open reading frames on the 140-MDa plasmid pRmeGR4b of Sinorhizobium meliloti strain GR4. Termed dapA and dapB, these encode products which show significant sequence similarities to DapA and DapB proteins, respectively. Escherichia coli DAP auxotrophs (dapA and dapB mutants) could be complemented with the pRmeGR4b dapA and dapB genes, indicating that these genes code for functional dihydrodipicolinate synthase and dihydrodipicolinate reductase, respectively. Reverse-transcriptase PCR analyses and beta-galactosidase assays using transcriptional dapA-lacZ and dapB-lacZ fusions suggest that these genes are constitutively expressed in S. meliloti. The dapA and dapB genes are not widely distributed in S. meliloti and appear to be specific for strains carrying pRmeGR4b-type plasmids.