The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

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.     

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     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.     

Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

Summary We studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO ⁺ G ⁺ ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when grown with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO ⁺ strain. The valine sensitivity of E. coli K-12 is a consequence of this regulatory pattern.     

Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12

Summary A strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants.The strain carrying the ilv0603 mutation is resistant to valine inhibition (Val^r) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Val^r acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Val^r acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Val^r peak is missing in a strain carrying an ilvG (amber) mutation.     

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.     

Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I₅₀ values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.     

Mutant of Escherichia coli K-12 Missing Acetolactate Synthase Activity

A mutant requiring isoleucine and valine for growth, because of the absence of acetolactate synthase activity, has been isolated. At least one of three different genes (ilvG, ilvB, ilvI) is required for the expression of acetolactate synthase activity, thus suggesting the presence of three different acetolactate synthase isoenzymes.     

Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl

Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10- to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism.     

The existence of three types of acetohydroxy acid synthetase in an isoleucine-requiring mutant of Aerobacter aerogenes.

The synthesis of the three types of acetolactate synthase (EC 4.1.3.18) which are responsible for the biosynthesis os isoleucine and valine, was observed in Aerobacter aerogenes I-12, an isoleucine-requiring mutant, when grown on the four kinds of media. When the cells were grown on isoleucine-rich medium, acetolactate synthase sensitive to feedback inhibition and having an optimum pH at 8.0 was formed. By increasing the amount of potassium phosphate in the medium, the catabolite repression of the enzyme having an optimum pH at 6.0 and which is insensitive to feedback inhibition, was released. In contrast, acetolactate synthase having an optimum pH at 8.0 and insensitive to feedback inhibition was formd when isoleucine was limited, irrespective of phosphate concentrations. Two insensitive enzymes were not regulated by isoleucine, leucine and valine, although sensitive pH 8.0 enzyme was repressed by them. Thus, it may be assumed that the synthesis of insensitive pH 8.0 enzyme were repressed by limiting the amount of isoleucine is still open.     

Valine-Resistant Escherichia coli K-12 Strains with Mutations in the ilvB Operon

Escherichia coli K-12 mutants resistant to growth inhibition by valine were isolated. These strains contained mutations in the ilvB operon effecting either the regulation of acetohydroxy acid synthase I or the sensitivity of the enzyme to end product inhibition by valine.     

Lack of Cross-Resistance of Imidazolinone-Resistant Cell Lines of Datura innoxia P. Mill. to Chlorsulfuron : Evidence for Separable Sites of Action on the Target Enzyme

Two cell lines of Datura innoxia resistant to two imidazolinone herbicides, imazapyr and imazaquin, were isolated from mutagenized, predominantly haploid cell suspension cultures. Both of the resistant variants were >1000-fold more resistant than the wild-type to the two imidazolinones. The variant resistant to imazapyr showed cross-resistance to imazaquin and vice versa, but no cross-resistance to a structurally different inhibitor, chlorsulfuron, a sulfonylurea herbicide, was observed. The target enzyme, acetolactate synthase, extracted from imidazolinone-resistant cell lines was not inhibited by imazapyr or imazaquin but was sensitive to chlorsulfuron indicating separable sites of action for these inhibitors. The variation in resistance and cross-resistance of chlorsulfuron-resistant (PK Saxena, J King [1988] Plant Physiol 86: 863-867) and imidazolinone-resistant cell lines of Datura innoxia demonstrates the possibility of separate mutations of acetolactate synthase gene resulting in specific phenotypes.     

Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in acetohydroxyacid synthase I function in vivo.

Mutations in Escherichia coli genes cpxA and cpxB together cause a temperature-sensitive defect in isoleucine and valine syntheses that is related specifically to acetohydroxyacid synthase I. This enzyme catalyzes the first pair of homologous reactions required for the synthesis of these two amino acids. At both permissive and nonpermissive temperatures, mutant cells containing ilvB (the structural gene for acetohydroxyacid synthase I) cloned in a derivative of plasmid pBR322 synthesized comparable amounts of ilvB mRNA and contained several times the enzyme activity normally required to sustain exponential growth, yet these cells remained temperature sensitive for growth in the absence of isoleucine and valine. These observations suggest that the primary effect of the cpx mutations is to block enzyme function in vivo. The enzyme was unstable in mutant cells at growth temperatures above 37 degrees C, but this instability appeared to be a secondary effect on the cpx mutations.     

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.     

Effects of deletion and insertion mutations in the ilvM gene of Escherichia coli.

A plasmid was constructed that carried the ilvG and ilvM genes and the associated promoter and leader regions derived from the K-12 strain of Escherichia coli. The ilvG gene contained a + 1 frameshift mutation that enabled the plasmid to specify acetohydroxyacid synthase II. The plasmid was modified by deletions in the terminus of and within the ilvM gene and by insertions into the ilvM gene. The effects of these modifications on the phenotypes of the plasmids were examined in a host strain that lacked all three isozymes of acetohydroxyacid synthase. Most of the ilvM mutant plasmids so obtained permitted growth of the host strain in the absence of isoleucine but not in the absence of valine. Growth in the presence of valine, however, was very slow. No significant acetohydroxyacid synthase activity could be detected even when the cells were grown in a valine-supplemented minimal medium. It thus appears that, at most, only a very low level of acetohydroxyacid synthase activity occurred with ilvG in the absence of ilvM and that low activity was more effective for acetohydroxy butyrate formation than for acetolactate formation. The ilvM gene product could be formed under the control of the lac promoter in the presence of a plasmid that carried an in-frame gene fusion between lacZ and the downstream portion of ilvG. Extracts from the host strain that contained such an IlvG(-)-IlvM+ plasmid could be combined with extracts from cells that contained one of the IlvG+-IlvM- plasmids to yield acetohydroxyacid synthase activity. Thus, the ilvM and ilvG genes could be expressed independently of each other.     

A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS.     

Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in isoleucine and valine syntheses.

Mutations in two chromosomal genes of Escherichia coli, cpxA and cpxB, produced a temperature-sensitive growth defect that was remedied specifically by the addition of isoleucine and valine to the minimal medium. This auxotrophy was manifested only when the medium contained exogenous leucine, suggesting that mutant cells fail to elaborate active acetohydroxy acid synthase, isozyme I. In the presence of leucine, this enzyme was required to catalyze the first reaction common to the biosynthesis of isoleucine and valine. Measurements of enzyme activity in crude extracts showed that mutant cells were seven- to eightfold deficient in active isozyme I when the cells were grown in the presence of leucine. When grown in the absence of leucine, mutant cells contained more acetohydroxy acid synthase activity. We attribute this activity to isozyme III, the product of the ilvHI genes, which are derepressed in the absence of exogenous leucine. The cpxA and cpxB mutations appear to affect the production of active isozyme I, rather than its activity, since (i) neither the cpxA nor the cpxB gene mapped near the structural gene for isozyme I (ilvB), (ii) the growth of mutant cells shifted from the permissive (34 degrees C) to the nonpermissive (41 degrees C) temperature did not immediately cease, but declined gradually over a period corresponding to several normal generation times, and (iii) the enzyme from mutant cells grown at 34 degrees C was as stable at 41 degrees C as the enzyme from cpx+ cells.