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 -ketobutyrate (KB) 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 KB 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 KB in place of -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 KB is that the ratio between intracellular KB and -ketoisovalerate is expected to be high in the first case, but not necessarily in the second.Abbreviations AHAS acetohydroxy acid synthase - BCAA branched-chain amino acids - IM imidazolinone - KB -keto-butyrate - SMM sulfometuron methyl - SU sulfonyl urea This research was supported in part by grant 338/92 from the Israel Science Foundation. It was also supported by the Lily and Sidney Oelbaum Chair in Applied Biochemistry, of which D.M.C. is the incumbent.     

Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.     

Acetolactate synthase inhibiting herbicides bind to the regulatory site

Acetolactate synthase from spontaneous mutants of tobacco (Nicotiana tabacum; KS-43 and SK-53) and cotton (Gossypium hirsutum; PS-3, PSH-91, and DO-2) selected in tissue culture for resistance to a triazolopyrimidine sulfonanilide showed varying degrees of insensitivity to feedback inhibitor(s) valine and/or leucine. A similar feature was evident in the enzyme isolated from chlorsulfuron-resistant weed biotypes, Kochia scoparia and Stellaria media. Dual inhibition analyses of triazolopyrimidine sulfonanilide, thifensulfuron, and imazethapyr versus feedback inhibitor leucine revealed that the three herbicides were competitive with the amino acid for binding to acetolactate synthase from wild-type cotton cultures. Acetolactate synthase inhibiting herbicides may bind to the regulatory site on the enzyme.     

INTRASPECIFIC TRANSFER OF HERBICIDE RESISTANCE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYCEAE) VIA PROTOPLAST FUSION

Stable progeny doubly resistant to the herbicides sulfometuron methyl (SMM) and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (DCMU) were obtained at a frequency of 2% on fusion of protoplasts derived from mutants of Porphyridium sp. (UTEX 637) that were resistant only to SMM (strain SMR) or DCMU (strain DC-2). In the presence of both herbicides, only the fusion progeny could grow; both parental mutants were inhibited. In the absence of SMM, the activity of acetohydroxy acid synthase (AHAS) in the wild-type strain was similar to that in DC-2, exceeding that of SMR by up to 4.5-fold. AHAS activities of all fusion progeny were lower than those of the wild-type strain and DC-2 but higher than that of SMR. In the presence of SMM, AHAS activities of all tested fusion progeny ranged between those of the two parental mutants. This result indicates that both types of AHAS, the type resistant to SMM and the sensitive type, originating from SMR and DC-2, respectively, were expressed in the fusion progeny. In the presence of DCMU, the photosynthetic activity of SMR was completely inhibited, whereas that of DC-2 was unaffected. The photosynthetic activity of the fusion progeny in the presence of DCMU was slightly lower than that of DC-2. Both the cell volume and the DNA content of the fusion progeny were similar to those of the parents. However, the genetic nature of the fusion products has not yet been elucidated. To the best of our knowledge, this is the first report on transfer of herbicide resistance via protoplast fusion in algae.     

AN HERBICIDE (SULFOMETURON METHYL) RESISTANT MUTANT IN PORPHYRIDIUM (RHODOPHYTA)1

The unicellular red alga Porphyridium sp. is, like many bacteria, fungi and higher plants, sensitive to the sulfonylurea herbicide sulfometuron methyl (SMM). However, the minimal inhibitory concentration for SMM in Porphyridium (55 μM) is higher than in green plants. We isolated a spontaneous SMM-resistant mutant SMR1 of Porphyridium sp. by use of a double-layered agar technique. The mutation frequency and the similarity of the mutant's morphology and growth patterns to the parent strain strongly suggest that SMR1 is a single gene mutation. The activity of the enzyme acetohydroxy acid synthase (AHAS) in crude extracts of the mutant SMR1 is at least two orders of magnitude less sensitive to SMM than that of the parent strain, which indicates that AHAS is the target of SMM (as has been shown in a variety of organisms). We propose that such a mutation, the first isolated in a unicellular rhodophyte, would be a useful marker for genetic studies of Porphyridium. It may also be useful for maintaining unialgal cultures in large scale open ponds.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 TrpLeu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.     

Acetohydroxy Acid Synthase Activity in Chlorella emersonii under Auto- and Heterotrophic Growth Conditions

Acetohydroxyacid synthase (AHAS) activity was studied in the green unicellular alga Chlorella emersonii. This activity and its regulation was compared in the algae grown autotrophically and heterotrophically on glucose in the dark. No evidence for the existence of more than one enzyme was found. The activity in crude extracts from either heterotrophically or autotrophically grown cells showed a K_m for pyruvate of 9 millimolar, a 22-fold preference for 2-ketobutyrate over pyruvate as the second substrate, 50% inhibition by 0.5 millimolar valine, and 50% inhibition by 0.3 micromolar sulfometuron methyl (SMM). Spontaneous mutants of the alga resistant to SMM were isolated, which appeared to be single gene mutants containing SMM-resistant AHAS activity. Hence, AHAS appears to be the sole direct target site of SMM in C. emersonii. The fact that the mutants had equivalent SMM resistance under auto- and heterotrophic conditions further supports the conclusion that the same enzyme functions under both physiological regimes. The addition of valine and isoleucine leads to partial relief of SMM inhibition of biomass increase, but not of SMM inhibition of cell division.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched‐chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over‐expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE‐TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map‐based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant‐containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.     

Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana

We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5 end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3 end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K.Abbreviations ALS acetolactate synthase - EMS ethylmethane sulfonate - POB pyrimidyl-oxy-benzoate The authors thank Mr. David Williams for his expert technical assistance and Mr. Dennis Dyck for help in preparing the figures.     

Biochemical evidence for multiple forms of acetohydroxy acid synthase in Spirulina platensis

Two isoforms of acetohydroxy acid synthase (AHAS), the first enzyme of the branched-chain amino acids biosynthetic pathway, were detected in cell-free extracts of the cyanobacterium Spirulina platensis and separated both by ion-exchange chromatography and by hydrophobic interaction. Several biochemical properties of the two putative isozymes were analysed and it was found that they differ for pH optimum, FAD requirement for both activity and stability, and for heat lability. The results were partially confirmed with the characterization of the enzyme extracted from a recombinant Escherichia coli strain transformed with one subcloned S. platensis coli strain transformed with one subcloned S. platensis AHAS gene. The approximate molecular mass of both AHAS activities, estimated by gel filtration, indicates that they are distinct isozymes and not different oligomeric species or aggregates of identical subunits.Abbreviations AHAS acetohydroxy acid synthase - DEAE cellulose diethylaminoethyl cellulose - DTT dithiothreitol - FAD flavin adenine dinucleotide - TPP thiamine pyrophosphate     

The distribution of acetohydroxyacid synthase in soil bacteria

Most bacteria possess the enzyme acetohydroxyacid synthase, which is used to produce branched-chain amino acids. Enteric bacteria contain several isozymes suited to different conditions, but the distribution of acetohydroxyacid synthase in soil bacteria is largely unknown. Growth experiments confirmed that Escherichia coli, Salmonella enterica serotype Typhimurium, and Enterobacter aerogenes contain isozymes of acetohydroxyacid synthase, allowing the bacteria to grow in the presence of valine (which causes feedback inhibition of AHAS I) or the sulfonylurea herbicide triasulfuron (which inhibits AHAS II) although a slight lag phase was observed in growth in the latter case. Several common soil isolates were inhibited by triasulfuron, but Pseudomonas fluorescens and Rhodococcus erythropolis were not inhibited by any combination of triasulfuron and valine. The extent of sulfonylurea-sensitive acetohydroxyacid synthase in soil was revealed when 21 out of 27 isolated bacteria in pure culture were inhibited by triasulfuron, the addition of isoleucine and/or valine reversing the effect in 19 cases. Primers were designed to target the genes encoding the large subunits (ilvB, ilvG and ilvI) of acetohydroxyacid synthase from available sequence data and a ∼355 bp fragment in Bacillus subtilis, Arthrobacter globiformis, E. coli and S. enterica was subsequently amplified. The primers were used to create a small clone library of sequences from an agricultural soil. Phylogenetic analysis revealed significant sequence variation, but all 19 amino acid sequences were most closely related to published large subunit acetohydroxyacid synthase amino acid sequences within several phyla including the Proteobacteria and Actinobacteria. The results suggested the majority of soil microorganisms contain only one functional acetohydroxyacid synthase enzyme sensitive to sulfonylurea herbicides.     

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 mol a.i. kg^–1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.Abbreviations AHAS acetohydroxyacid synthase - CETAB cetyltrimethylammonium bromide - ID₅₀ concentration causing 50% inhibition of enzyme activity - LD₅₀ concentration causing 50% decrease of growth constant value     

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase