Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.     

The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15.

The biosynthetic gene cluster of the myxochelin-type iron chelator was cloned from Stigmatella aurantiaca Sg a15 and characterized. This catecholate siderophore was only known from two other myxobacteria. The biosynthetic genes of 2,3-dihydroxybenzoic acid are located in the cluster (mxcC-mxcF). Two molecules of 2, 3-dihydroxybenzoic acid are activated and condensed with lysine in a unique way by a protein homologous to nonribosomal peptide synthetases (MxcG). Inactivation of mxcG, which encodes an adenylation domain for lysine, results in a myxochelin negative mutant unable to grow under iron-limiting conditions. Growth could be restored by adding Fe3+, myxochelin A or B to the medium. Inactivation of mxcD leads to the same phenotype. A new type of reductive release from nonribosomal peptide synthetases of the 2, 3-dihydroxybenzoic acid bis-amide of lysine from MxcG, catalyzed by a protein domain with homology to NAD(P) binding sites, is discussed. The product of a gene, encoding a protein similar to glutamate-1-semialdehyde 2,1-aminomutases (mxcL), is assumed to transaminate the aldehyde that is proposed as an intermediate. Further genes encoding proteins homologous to typical iron utilization and iron uptake polypeptides are reported.     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.     

Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae results in a greatly reduced affinity for the herbicide glyphosate

The aroA gene of Klebsiella pneumoniae encoding the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, which is the target of the herbicide glyphosate, was cloned and sequenced from both the wild-type and the glyphosate-resistant mutant K. pneumoniae K1, which possesses a glyphosate-insensitive EPSP synthase. Both genes were expressed in Escherichia coli and were capable of complementing an auxotrophic aroA mutation. The transformed cells showed increased tolerance to glyphosate due to the overproduction of either the mutant or the wild type EPSP synthase. Nucleotide sequence analysis of the K. pneumoniae aroA gene indicated a protein-coding region of 427 amino acids with a derived Mr for the EPSP synthase of 45,976. Comparison of the two aroA alleles showed a single base change resulting in a substitution of Gly-96 to Ala in the deduced amino acid sequence. By comparison with other known EPSP synthase sequences the mutation was shown to be located in a highly conserved region, indicating that this region is essential for the binding of the herbicide glyphosate.     

Isolation of Pseudomonas aurantiaca strains capable of overproduction of phenazine antibiotics

N-methyl-N'-nitro-N-nitrosoguanidine (NH)-induced mutagenesis with subsequent selection for resistance to toxic amino acid analogues (azaserine, m-fluoro-DL-phenylalanine, and 6-diazo-5-oxo-L-norleucine) was applied to Pseudomonas aurantiaca B-162. The resulting strains produced phenazine antibiotics three times more efficiently than the wild type strain and ten times more efficiently than the known pseudomonad strains. Overproduction of phenazine antibiotics was shown to result either from deregulation of 3-deoxi-D-arabinohepulosonate-7-phosphate synthase (DAHP synthase), the key enzyme of the aromatic pathway (removal of inhibition by phenylalanine, tyrosine, and phenazine), or overproduction of N-hexanoyl homoserine lactone, the regulatory molecule of positive control of cellular metabolism (QS system).     

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss‐of‐function mutation in cytochrome P450 72A69

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3‐dihydro‐2,5‐dihydroxy‐6‐methyl‐4H‐pyran‐4‐one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized β‐amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map‐based cloning and characterization of the gene Sg‐5, which is responsible for SA biosynthesis. The naturally occurring sg‐5 mutant lacks group A saponins and has a loss‐of‐function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg‐5 mutant, indicating that Glyma15g39090 corresponds to Sg‐5. Endogenous levels of DDMP saponins were higher in the sg‐5 mutant than in the wild‐type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg‐5 paralogs in a genomic region syntenic to the soybean Sg‐5 region. However, SA did not accumulate in common bean samples, suggesting that Sg‐5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg‐5.     

Substrate Ambiguity of 3-Deoxy-d-manno-Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae in the Context of Its Membership in a Protein Family Containing a Subset of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases

3-Deoxy-d-manno-octulosonate 8-phosphate (KDOP) synthase and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing reactions. The genome of Neisseria gonorrhoeae contains one gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the two nonhomologous DAHP synthase families known, the N. gonorrhoeae protein belongs to the family I assemblage. KDOP synthase exhibited an ability to replace arabinose-5-P with either erythrose-4-P or ribose-5-P as alternative substrates. The results of periodate oxidation studies suggested that the product formed by KDOP synthase with erythrose-4-P as the substrate was 3-deoxy-d-ribo-heptulosonate 7-P, an isomer of DAHP. As expected, this product was not utilized as a substrate by dehydroquinate synthase. The significance of the ability of KDOP synthase to substitute erythrose-4-P for arabinose-5-P is (i) recognition of the possibility that the KDOP synthase might otherwise be mistaken for a species of DAHP synthase and (ii) the possibility that the broad-specificity type of KDOP synthase might be a relatively vulnerable target for antimicrobial agents which mimic the normal substrates. An analysis of sequences in the database indicates that the family I group of DAHP synthase has a previously unrecognized membership which includes the KDOP synthases. The KDOP synthases fall into a subfamily grouping which includes a small group of DAHP synthases. Thus, family I DAHP synthases separate into two subfamilies, one of which includes the KDOP synthases. The two subfamilies appear to have diverged prior to the acquisition of allosteric-control mechanisms for DAHP synthases. These allosteric control specificities are highly diverse and correlate with the presence of N-terminal extensions which lack homology with one another.3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) and 3-deoxy-d-manno-octulosonate 8-phosphate (KDOP) are analogous seven- and eight-carbon 2-keto-3-deoxy sugars that are synthesized by enzymes which belong to functionally unrelated pathways. DAHP synthase forms DAHP as the acyclic precursor of the aromatic amino acids in bacteria, lower eukaryotes, and plants (3); KDOP synthase is best known for its role in the formation of KDOP as a critical component of the lipopolysaccharide of gram-negative bacteria (37), but its distribution in nature has recently been recognized to be broader (13). Both enzymes catalyze an overall condensation of phosphoenolpyruvate (PEP) with an aldose, i.e., erythrose-4-phosphate (E4P) in the case of DAHP synthase and arabinose-5-phosphate (A5P) in the case of KDOP synthase. The reactions are irreversible and are not aldol-type condensations, which unfortunately has been implied by the Enzyme Commission naming that has been recommended for DAHP synthase.As might be expected from the close structural relationship of A5P and E4P, the reactions are strikingly similar. This similarity is reflected at the level of mechanistic detail (see reference 16 and references therein). DAHP synthase and KDOP synthase, along with enolpyruvoylshikimate 3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvoyl transferase, comprise a small class of PEP-utilizing enzymes that catalyze C—O bond cleavage with respect to the release of P_i from PEP (1, 27). This contrasts with the more familiar nucleophilic attack at the phosphorous atom of PEP that results in P—O bond cleavage by the action of enzymes such as pyruvate kinase (25), PEP carboxylase (34), and PEP carboxykinase (8).In classical studies with Escherichia coli, DAHP synthase (44, 45) and KDOP synthase (41) are specific for E4P and A5P, respectively. In contrast, we found that the KDOP synthase of Neisseria gonorrhoeae possessed the ability to utilize E4P in place of A5P. We addressed the question of whether KDOP synthase of N. gonorrhoeae in the presence of E4P and PEP was able to form DAHP, in which case it would also have the potential to function as a DAHP synthase. The time-dependent cleavage of the product was investigated by the periodate-oxidation-thiobarbituric acid (TBA) assay, and these results allow some speculation on the stereospecific course of the reaction in comparison with the reaction of DAHP synthase.     

Metabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90.

Myxobacterial strains producing polyketides (PKs) assumed to be biosynthesized by a type I polyketide synthase (PKS) were analysed. Myxobacteria also produce a variety of polypeptides (PP) and PKs with incorporated amino acids ('mixed PK-PP'). In order to be able to identify the biosynthetic gene clusters for these metabolites a PCR based approach has been developed to clone ketosynthase (KS) domains of PKS genes from these organisms. Conserved regions of peptide synthetases of the non-ribosomal type (NRPS) were also amplified via PCR. KS fragments from Stigmatella aurantiaca Sg a15 were used for chromosomal gene inactivation experiments resulting in a series of mutants including such that were unable to produce stigmatellins and myxalamids. A NRPS fragment and PKS fragments from Sorangium cellulosum So ce90 were used to identify cosmids hybridizing with both types of probes from a genomic library. Both a NRPS and a PKS fragment were cloned and sequenced from a relatively short restriction fragment of one of these cosmids. The method described here should be very useful to clone and identify PKS, NRPS and mixed PKS-NRPS from myxobacteria in general and thereby open opportunities to use the biochemical diversity of these bacteria for genetic engineering and combinatorial biosynthesis.     

The cloning and nucleotide sequence of a Corynebacterium glutamicum 3-deoxy-d- arabinoheptulosonate-7-phosphate synthase gene

Abstract The aro gene of Corynebacterium glutamucum CCRC 18310 encoding 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase was isolated by complementation of a DAHP synthase-deficient mutant of Escherichia coli AB3257. The specific activity of DAHP synthase was increased four-fold in a C. glutamicum strain harboring the cloned aro gene. The complete nucleotide sequence of the aro gene and 5' and 3' flanking regions has been determined. The sequence contained an open reading frame of 368 codons, from which a protein with a molecular mass of 39 340 Da could be predicted. The deduced amino acid sequence shows high identity with the aro gene products of E. coli and Salmonella typhimurium .     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield.

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.     

Translocation of 3-deoxy-<Emphasis Type="SmallCaps">D</Emphasis><Emphasis Type="Italic">-arabino</Emphasis>-heptulosonate 7-phosphate synthase precursor into isolated chloroplasts

A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) from potato (Solanum tuberosum L.) presumably specifies a chloroplast transit sequence near its 5'-end. In order to show the function of this transit sequence, we constructed a plasmid that contains the entire coding region of the cDNA downstream from a T7 promoter. Using this plasmid as template, DAHP synthase mRNA was synthesized in vitro with T7 RNA polymerase. The resulting mRNA served as template for the in vitro synthesis of a 59-kDa polypeptide. This translation product was identified as the DAHP synthase precursor by immunoprecipitation with a monospecific polyclonal antibody raised against pure tuber DAHP synthase and by radiosequencing of the [(3)H]leucine-labeled translation product. Incubation of the 59-kDa polypeptide with isolated spinach (Spinacia oleracea L.) chloroplasts resulted in a 53-kDa polypeptide that was resistant to protease treatment. Fractionation of chloroplasts, reisolated after import, showed the mature DAHP synthase in the stroma fraction. Incubation of the 59-kDa polypeptide with a chloroplast precursor-processing enzyme cleaved the precursor between Ser49 and Ala50, generating a mature DAHP synthase of 489 residues. The uptake of the DAHP synthase precursor into isolated chloroplasts was inhibited by anti-DAHP synthase, and the precursor was not processed cotranslationally by canine microsomal membranes. We conclude that the transit sequence is able to direct DAHP synthase into chloroplasts.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II.

Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium. Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with Tn10. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The Tn10 insertion in strain SMS409 was mapped by conjugation and transduction to the region between aroA and pncB at 20 min on the chromosome of S. typhimurium; this location is similar to the genetic location of aspC in Escherichia coli. The specific activity of the aspC product, aspartate aminotransferase, was severely reduced in strain SMS409. This indicated that the Tn10 insertion in strain SMS409 inactivated aspC. An aspC mutant of E. coli was also inhibited by either sulfometuron methyl or tyrosine. We present a hypothesis which relates the observed alpha-ketobutyrate accumulation in sulfometuron methyl-inhibited cultures of strain SMS409 to aspartate starvation.     

Differential expression of DAHP synthase and chorismate mutase isozymes during rhizogenesis of Brassica juncea cultured in vitro

The regulatory patterns of two of the enzymes of the shikimate pathway. 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase or DS. EC 4. 1. 2. 15) and chorismate motase (CM, EC 5. 4. 99. 5), were investigated using in vitro cultures of Brassica juncea at two stages, viz. undifferentiated, proliferating callus and the root-forming callus. Our studies revealed the presence of the two isozymes of DAHP synthase, DS-Mn and DS-Co. in undifferentiated callus. However, during the rhizogenesis of the callus DS-Mn was absent. Similarly, for chorismate mutase, whereas both the isozymes CM-1 and CM-2 were present in undifferentiated callus only CM-2 was detected at rhizogenesis. The possible involvement of these isozymes in callus growth and rhizogenesis is discussed.