AHAS Activity Research Articles

Multiple Effects of L-Leucine in Escherichia coli Lead to L-Leucine-Sensitive Growth in the Absence of Unphosphorylated PtsN.

In E. coli K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K+. This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α-ketobutyrate level mediate elevation of ilvBN expression and alleviate the LeuS. The requirement of unphospho-PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function.

Occurrence and mechanism of target-site resistance to bensulfuron-methyl in Monochoria korsakowii from China

Monochoria korsakowii is an increasingly significant threat to rice production across China, particularly in Liaoning province. Few studies have reported herbicide resistance in M. korsakowii, and resistance status and mechanisms are poorly understood. Here, thirty field populations of M. korsakowii were collected from 11 rice-growing regions of Liaoning, and 97% of populations had evolved resistance to bensulfuron-methyl (BM), with majority (24 of 28) showing high resistance levels (RI > 10). The first in-depth analysis of molecular features of AHAS1 and AHAS2 in BM-resistant populations showed that four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified. Notably, novel double Pro197Ser mutations co-occurred in both AHAS1 and AHAS2 in the most resistant line LN-20. Furthermore, resistant mutants were used to investigate the effect of Pro197 mutations on AHAS functionality, binding modes, gene expression and cross-resistance in M. korsakowii. All the detected Pro197 mutations considerably reduced in vitro AHAS sensitivity to BM by weakening hydrogen bonds and hydrophobic interactions in the predicted BM-AHAS complexes, especially the double Pro197Ser mutations. This novel resistance mutation combination slightly impacted the extractable AHAS activity, and increased the affinity and catalytic rate of pyruvate. Also, the AHAS expression level was significantly up-regulated. Moreover, all mutations provided resistance only to other sulfonylureas herbicides but not triazolopyrimidine or pyrimidinyl-benzoates herbicides. In conclusion, bensulfuron-methyl resistance in M. korsakowii was grim in Liaoning, China, and amino acid mutations on AHAS isozymes were the primary resistance mechanism. Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. Thus, this work deepens our understanding of resistance status and mechanisms of M. korsakowii.

Cultivar variation for imazamox resistance in wheat (Triticum aestivum L.): Insights into enzymatic assays for early selection

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the target site of several herbicide classes including imidazolinones. Imidazolinone resistance in wheat is conferred by two major genes AhasL-D1 and AhasL-B1. The objective of this work was to evaluate the in vitro and in vivo AHAS activity and plant growth in response to imazamox of nine wheat cultivars. Dose-response curves for two-gene resistant cultivars were significantly different from the single-gene resistant and susceptible cultivars in the in vitro AHAS assay. Resistance levels at the in vivo AHAS and whole-plant assays for resistant cultivars were >10-fold higher than susceptible cultivars. Moreover, in vivo dose-response curves showed differences among cultivars with the same number of resistance genes. It was concluded that in the in vitro AHAS assay cultivar variability was due to differences in target-site sensitivity while the in vivo AHAS assay reflected the resistance at whole-plant level. Both in vitro and in vivo AHAS dose-response curves could be useful tools when exploring mechanisms involved in imidazolinone resistance in different wheat genetic backgrounds and for the selection of higher resistant genotypes.

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower.

Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose-response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids. © 2018 Society of Chemical Industry.

Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis

Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.

An in vitro mutagenesis protocol for the production of sugarcane tolerant to the herbicide imazapyr

A protocol was developed to induce and identify imazapyr tolerance in sugarcane, which involved induction of somaclonal variation via exposure to 8 or 16 mM ethyl methanesulfonate for 4 h, followed by a stepwise increase in imazapyr concentration in the medium from 0.08 to 0.16 μM. The regenerated plantlets were then acclimatized for 3 mo after which they were sprayed with 182 g a.i. ha−1 imazapyr, and the above-ground biomass was determined after 47 d. Following a 1-mo waiting period for the putative tolerant plants to regrow, acetohydroxyacid synthase (AHAS; EC 2.2.1.6) enzyme assays of the plants that survived and showed a normal growth pattern were undertaken. Based on the enzymatic I 50 values, three imazapyr-tolerant genotypes were identified with an AHAS activity of 2.8 to 4.0 times that in sensitive sugarcane plants.

Inhibitors of Bacillus anthracis acetohydroxyacid synthase

Twelve compounds (100 μM) had a predicted log P between −0.5 and −3.0 and inhibited AHAS activity by >40%. CE, IQ, and SMM showed dose-dependent inhibition of Bacillus anthracis AHAS (BAntx AHAS) with IC 50 values of 7.01 ± 0.81, 6.97 ± 0.44, and 10.02 ± 1.42 μM, respectively. CE and IQ were noncompetitive and uncompetitive inhibitors of BAntx AHAS with K ii values of 3.77 and 2.02, respectively. The order of potency towards BAntx AHAS was CE > PSE > MSM > TSM > SMM for sulfonylureas and IQ > Imazapic > IP for imidazolinones. The most active compound, CE, showed the lowest total interaction energy and highest MolDock score of −140.054 kcal/mol and −141.52, respectively. Three hydrogen bonds were identified between BAntx AHAS and CE. AHAS appears to be a promising potential target for new anti-anthracis drugs.

New industrial brewing yeast strains with ILV2 disruption and LSD1 expression

New industrial brewing yeast strains, free of vector sequences and drug-resistance genes, were constructed by disrupting α-acetohydroxyacid synthase (AHAS) gene ( ILV2) and introducing Lipomyces starkeyi dextranase (DEX) gene ( LSD1) as a selective marker. The resulting recombinant strains can survive on YNB minimal medium plate with dextran T-70 as sole carbon source and showed lower AHAS activity. Fermentation test with recombinant strains in 500 ml conical flask confirmed DEX activity and lower AHAS activity compared with their host strain. Moreover, the fermentative performance of recombinant strains T1 and Q9 was better than their host, and the residual sugar content was reduced by 20–25% in fermented wort with recombinant strains compared to their host, too.

Metabolic response of Chlorella emersonii to the herbicide sulfometuron methyl

Although it is clear that acetohydroxy acid synthase (AHAS; EC 4.1.3.18) is the target for sulfonylurea herbicides such as sulfometuron methyl (SMM), there is considerable uncertainty as to the mechanism(s) by which inhibition of AHAS inhibits or kills cells. We have further studied the mode of action of SMM, and its effects on metabolism and physiology in the unicellular green alga Chlorella emersonii var. emersonii. Addition of SMM to cells synchronized to a cycle of 16 h light-8 h dark showed that they were very sensitive to SMM toxicity in the first 16 h of the cell cycle, during which cell mass, protein and DNA increased. The increase in protein, DNA and chlorophyll was halted rapidly after SMM addition. Sulfometuron methyl prevented cell division even if added late in the light stages, when most of the protein and DNA were already synthesized, but did not affect cell division and autospore release if added after protein and DNA synthesis were complete. This suggests that SMM interferes with processes involved in preparation for division, beyond what would be expected if the cells were starved of the branched-chain amino acids needed as precursors for synthesis of proteins in general. The accumulation of α-ketobutyrate (αKB) in the cells in response to addition of SMM, and its possible role in the growth inhibition, was also investigated (in continually illuminated cultures). Intracellular αKB accumulated rapidly within 30 min of SMM addition, but declined nearly to basal levels in several hours. This paralleled the decrease and subsequent recovery of extractable AHAS activity. Despite this, growth of the algal culture did not recover. We suggest that metabolites formed by misincorporation of αKB in place of α-ketoisovalerate (e.g., in the ketopantoate hydroxymethyl transferase reaction) might be responsible for the persistence of growth inhibition. We note that an important difference between the effect of SMM and that observed with externally added αKB is that the ratio between intracellular αKB and α-ketoisovalerate is expected to be high in the first case, but not necessarily in the second.

Regulation of acetohydroxyacid synthase activity levels in transgenic tobacco

The Brassica napus nuclear gene, AHAS3R, codes for a mutant chloroplast enzyme acetohydroxyacid synthase (AHAS, EC 4.1.3.18) that is resistant to inhibition by the sulfonylurea herbicide, chlorsulfuron. It was introduced into Nicotiana tabacum under control of the cauliflower mosaic virus 35S promoter and leader sequences. The relative mRNA levels of the AHAS3R gene and the resident tobacco AHAS genes, SURA and SURB, were determined by RNAase protection assays. The AHAS3R mRNA level varied by more than 6-fold among different transformants, depending on the position of insertion and on gene dosage. The expression level was generally consistant with the percentage of AHAS activity that was chlorsulfuron-resistant. In contrast, the specific activity of AHAS, which includes the resident tobacco AHAS and introduced AHAS3R activities, was shown to be independent of AHAS3R gene dosage, mRNA level and proportion of chlorsulfuron-resistant enzyme activity. These observations reveal the presence of a post-transcriptional mechanism that governs the total amount of AHAS activity in the transgenic plants. When AHAS3R mRNA levels were at their highest, SURA and SURB activities appeared to be completely replaced by AHAS3R activity.

Acetohydroxyacid Synthase from Cell Suspension Cultures of Isatis tinctoria l. and Ruta graveolens l.

Summary Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from cell suspension cultures of Isatis tinctoria (Cruciferae) and Ruta graveolens (Rutaceae). A combination of salt precipitation, gel filtration and ion exchange chromatography was used for partial purification. The apparent molecular masses of AHAS were Mr 82,000 and 85,000 for Isatis and Ruta, respectively. FAD was an absolute requirement for AHAS activity. The apparent Km values of Isatis-AHAS are the following ones: FAD 6,3 × 10−6 M; TPP 6,3 × 10−6 M; pyruvate 7 × 10−3, and 6 × 10−3 M (for Ruta-AHAS). Branched-chain amino acids and chlorsulfuron are feedback inhibitors for Isatis-AHAS but acetohydroxyacid synthase from Ruta is not sensitive to valine, leucine and isoleucine.

Resistance to the Sulfonylurea Herbicides Chlorsulfuron, Amidosulfuron, and DPX‐R9674 in Transgenic Flue‐Cured Tobacco

Only one herbicide is currently available for preemergence broadleaf weed control in flue‐cured tobacco (Nicotiana tabacum L.) grown in Canada. The high cost of registration, coupled with the small crop size, has resulted in few new products becoming available. Herbicide resistance introduced into tobacco by Agrobacterium‐mediated transformation may allow the use of products with existing or impending registrations. We used two new, low‐residual sulfonylurco herbicides: amidosulfuron (3.(4,6‐dimethoxyprymidin.2.yl)‐l‐(N‐methyl‐N‐methylsulfonyl‐aminosulfonylurea) and DPX‐R9674, which is a mixture of thifensulfuron (methyl‐3‐ [[[[(4‐methoxy‐6‐methyl‐ 1,3,5‐triazin‐2‐yl)amino] carbonyl] amino] sulfonyl]‐2‐thiophenecarboxylate) and tribenuron (methyl 2[[[[N‐4‐methoxy‐6‐methyl‐l,3,5‐triazin‐2‐yl)methylamino] carbonyl] amino] sulfonyl] benzoate). These two and chlorsulfuron (2.chloro‐N‐[(4‐methoxy‐6.methyl‐l,3,5‐triazin‐2‐yl) aminocarbonyl] benzenesulfonamide) were applied to two transgenic tobacco genotypes harboring the csrl‐1 gene for chlorsulfuron resistance and compared with an untransformed control. Our purpose was to determine if transgenic seedlings were resistant to DPX‐R9674 and amidosulfuron. The experiment was a factorial in a completely randomized design with 25 replications. The three herbicides were applied to the transgenic and control seedlings at three rates. The transgenic seedlings had significantly higher leaf area, top dry weight, and root dry weight than the untransformed control when sprayed with any of the three herbicides. Seedlings were highly resistant to amidosulfuron and chlorsulfuron. Resistance to DPX‐R9674 in the transgenic seedlings was minimal, which was unexpected, considering that an analysis of AHAS activity revealed high levels of cross‐resistance to chlorsulfuron, DPX.R9674, and amidosulfuron. It is possible that DPX‐R9674 is metabolized into products that are herbicidally active at different AHAS binding sites. One of the transgenic lines was more resistant to herbicide application than the other indicating that selection for maximum gene expression among transgenic lines is a necessary part of transgenic cultivar development. It was concluded that DPX‐R9674 would not be suitable for use with transgenic crops harboring the csrl‐1 gene for chlorsulfuron resistance. The other lowresidual sulfonylurea, amidosulfuron, was more promising.

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In cross-reactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Western blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of cross-reactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species.

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during isoleucine formation from ?-hydroxybutyric acid

When Corynebacterium glutamicum ATCC 14310 (leu-) was cultured with 200 mg/l leucine and 150 mM α-hydroxybutyric acid the acetohydroxy acid synthase activity was increased to 0.17 U/mg as compared to 0.03 U/mg in the wildtype. This increase was a combined effect of the limiting amounts of leucine added, together with an apparent additional internal leucine/valine shortage resulting from accumulated α-ketobutyric acid (5 mM) and the kinetic characteristics of the acetohydroxy acid synthase. The increase in the specific AHAS activity by the appropriate amino acid limitation resulted in an increased isoleucine yield of 71 mmol/l as compared to 27 mmol/l obtained under non-limiting conditions.

Regulation of acetohydroxy acid synthase activities in adenyl cyclase-deficient strains of Escherichia coli K-12.

Previous findings suggested that cyclic AMP was involved in the regulation of ilvB(AHASI) only and that ilvG (AHASII) and ilvHI (AHASIII) were not controlled by this nucleotide. In this study, derepression patterns of total AHAS activities (ilvB and ilvHI) in adenyl cyclase-negative strains (i.e. cya-) were substantially reduced as contrasted with AHAS activity observed for cya+ strains. Further, the parental strains (cya+) consistently exhibited higher levels of AHAS activity than mutant strains (cya-) during carbon and energy downshifts. Other data suggested that the valine derepression signal could not override the necessity for cya gene product to yield maximal derepression of AHAS gene activities. Cyclic AMP stimulated AHAS gene activities under both in vivo and in vitro assay conditions. Thus, these data provide evidence for an absolute requirement of cAMP for maximal expression of the genes encoding for AHAS activities of E. coli K-12.

Metabolic basis for the isoleucine, pantothenate or methionine requirement of ilvG strains of Salmonella typhimurium.

Salmonella typhimurium strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated alpha-ketobutyrate and, to a lesser extent, alpha-aminobutyrate. We found that alpha-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, alpha-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced alpha-ketobutyrate could be mimicked in wild-type S. typhimurium by exogenously supplied alpha-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of alpha-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the alpha-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of alpha-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of alpha-keto-beta-methylvalerate and aminobutyrate from isoleucine and alpha-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of alpha-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic ilvG mutant strains.

Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: Unique regulation modulated by multiple genetic sites

Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Valr). The Valr isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHASr), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHASr phenotype were found to be cotransducible with glyA. However, no mutations causing a Valr phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Valr linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHASs) and AHASr in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHASs, only, in the mutant containing ilv-662. Further, both AHASs and AHASr in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHASr synthesis in the strain carrying ilv-660 and only partially repressed AHASr in the strain carrying ilv-662. Unexpectedly, AHASr synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.

NEW MAP LOCATION OF ilvO IN ESCHERICHIA COLI

Mutations in ilvO, the operator site for operon A of the ilv region of E. coli K-12, are reported to cause a cis dominant derepression of the operon A gene products and to map between ilvC and ilvA. Studies reported here demonstrate that the F25 episome which does not carry the region between ilvC and ilvA but only ilvE, ilvD and a part of ilvA, can transfer ilvO genetic material. Also, genes of operon A on the F25 episome respond normally to a derepression signal. It is proposed that ilvO is on the F25 episome. Five-point crosses demonstrate that mutations in ilvO map nearer to ilvE than to ilvA. Considering this and other evidence, it is proposed that the gene order in the ilv region is CADEO, not COADE, with transcription and translation in operon A being from ilvE to ilvA not ilvA to ilvE. It is confirmed that mutations in ilvO cause a cis dominant derepression of the operon A gene products but it is also noted that mutations in ilvO lead to the appearance of an AHAS activity more resistant to inhibition by valine than that of the present O(+) strain.