The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium

The sulfonylurea herbicide sulfometuron methyl inhibits the growth of several bacterial species. In the presence of L-valine, sulfometuron methyl inhibits Salmonella typhimurium, this inhibition can be reversed by L-isoleucine. Reversal of growth retardation by L-isoleucine, accumulation of guanosine 5'-diphosphate 3'-diphosphate (magic spot), and relA mutant hypersensitivity suggest sulfometuron methyl interference with branched-chain amino acid biosynthesis. Growth inhibition of S. typhimurium is mediated by sulfometuron methyl's inhibition of acetolactate synthase, the first common enzyme in the branched-chain amino acid biosynthetic pathway. Sulfometuron methyl exhibits slow-binding inhibition of acetolactate synthase isozyme II from S. typhimurium with an initial Ki of 660 +/- 60 nM and a final, steady-state Ki of 65 +/- 25 nM. Inhibition of acetolactate synthase by sulfometuron methyl is substantially more rapid (10 times) in the presence of pyruvate with a maximal first-order rate constant for conversion from initial to final steady-state inhibition of 0.25 +/- 0.07 min-1 (minimal half-time of 2.8 min). Mutants of S. typhimurium able to grow in the presence of sulfometuron methyl were obtained. They have acetolactate synthase activity that is insensitive to sulfometuron methyl because of mutations in or near ilvG, the structural gene for acetolactate synthase isozyme II.     

ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl.

The herbicide sulfometuron methyl is a potent inhibitor of the branched-chain amino acid biosynthetic enzyme acetolactate synthase (ALS) isolated from bacteria, fungi, and plants. However, it did not prevent growth of wild-type Salmonella typhimurium LT2 or Escherichia coli K-12. These species each contain two acetolactate synthase isozymes. Growth of S. typhimurium and E. coli mutants lacking ALS I was prevented by the herbicide, suggesting that activity of the remaining ALS isoenzyme (II or III, respectively) was stopped by sulfometuron methyl. Synthesis of ALS I requires either an relA function or an elevated cyclic AMP level. A relA mutant of S. typhimurium was inhibited by sulfometuron methyl on rich carbon sources that display a basal cyclic AMP level but not on poor carbon sources where the cyclic AMP concentration is elevated. When L-valine, which allosterically inhibits ALS I activity, was added, growth retardation of the relA- strain by sulfometuron methyl was observed on both poor and rich carbon sources. Enzymological analyses indicated that ALS I activities derived from both species were resistant to the herbicide. In contrast, activities of S. typhimurium ALS II and E. coli ALS III were abolished by sulfometuron methyl.     

Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains

Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.     

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.     

Toxic accumulation of alpha-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium.

Biochemical and genetic analyses of the bacterium Salmonella typhimurium suggest that accumulation of alpha-ketobutyrate partially mediates the herbicidal activity of acetolactate synthase inhibitors. Growth inhibition of wild-type bacteria by the herbicide sulfometuron methyl was prevented by supplementing the medium with isoleucine, an allosteric inhibitor of threonine deaminase-catalyzed synthesis of alpha-ketobutyrate. In contrast, isoleucine did not rescue the growth of a mutant containing a threonine deaminase unresponsive to isoleucine. Moreover, the hypersensitivity of seven Tn10 insertion mutants to growth inhibition by sulfometuron methyl and alpha-ketobutyrate correlated with their inability to convert alpha-ketobutyrate to less noxious metabolites. We propose that alpha-ketobutyrate accumulation is an important component of sulfonylurea and imidazolinone herbicide action.     

A slow-growing, ribosomal mutant of Salmonella typhimurium

Summary The mechanisms of S. typhimurium reversion from histidine dependence (his ^–) to histidine independence (his ⁺) were studied. Genetic and phenotypic characteristics of revertants induced by nitrosoguanidine were analyzed. Among them a class of slow-growing revertants was selected. It is found that all of these slow-growing revertants carry the original UGA nonsense mutation within the histidine operon. They are streptomycin sensitive and no specific suppressor(s) for UGA nonsense codon are demonstrable. The suppression takes place in the absence of conventional nonsense UGA suppressor(s). It is seemingly due to a ribosomal mutation which in turn is likely to produce ambiguity in the process of translation and which suppresses the UGA nonsense codon. The rate of both in vivo and in vitro protein synthesis is significantly reduced. The fact that streptomycin, at sublethal doses, reduced the growth rate of these mutants, probably because of the simultaneous burden of two ambiguity factors, suggests that the mutants described may be regarded as a kind of ram (ribosomal ambiguity) mutants with a his ^– sup ^–genotype. Their capacity to translate poly-U is reduced and in that respect they differ from ram mutants of Escherichia coli.     

Excretion of flagellin by a short-flagella mutant of Salmonella typhimurium.

A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (less than 0.1 micron long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.     

Characterization of a cold-sensitive hisW mutant of Salmonella typhimurium

Previous studies of hisW mutants of Salmonella typhimurium have led to the suggestion that such strains are defective in tRNA maturation. (J. E. Brenchley and J. Ingraham, J. Bacteriol. 114:528-536, 1973). In this study, we report that one hisW strain is defective in the accumulation of all stable RNA species. Polyacrylamide gel electrophoresis of radiolabeled RNA indicated tha at the nonpermissive temperature (20 degrees C) all stable RNa species in the cold-sensitive hisW3333 mutant were synthesized and rapidly degraded. We propose that the cold sensitivity of this strain is caused by such a restriction in stable RNA accumulation at low temperature. In vitro and in vivo studies demonstrated that the RNA degraded in this strain was synthesized de novo and was not preexisting RNA. Furthermore, physiological and genetic recovery from the cold-sensitive hisW phenotype resulted in relatively normal RNA synthesis and accumulation. Thus, the RNA alterations observed in this strain were not explained by defects in a tRNA modification enzyme. Rather, these findings suggest the existence of defective RNA processing and that a control mechanism for the overall synthesis or accumulation of stable RNA species is altered in the hisW3333 mutant.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Isolation of a trans-dominant histidase-negative mutant of Salmonella typhimurium

A mutation of Salmonella typhimurium was obtained that results in the failure of cells to synthesize the enzyme l-histidine ammonia-lyase (histidase). The mutation mapped within the hutH gene and in merodiploid strains was dominant over the wild-type allele. Extracts from cells bearing the trans-dominant histidase-negative allele were shown to contain material that reacts immunologically with antiserum against purified wild-type histidase. It is proposed that the trans-dominant allele results in the synthesis of defective histidase subunits that can combine with, and partially inactivate, wild-type histidase subunits. This subunit mixing presumably does occur, as the enzyme synthesized in a hybrid merodiploid strain is abnormally heat sensitive.     

Analysis of a FliM-FliN flagellar switch fusion mutant of Salmonella typhimurium.

In the course of an analysis of the three genes encoding the flagellar motor switch, we isolated a paralyzed mutant whose defect proved to be a 4-bp deletion of the ribosome binding sequence of the fliN switch gene (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810,1993). This sequence lies just before the 3' end of the coding sequence of the upstream fliM switch gene, in the same operon. This mutant readily gave rise to pseudorevertants which, though much less motile than the wild type, did exhibit significant swarming. One such pseudorevertant was found to contain a compensating frameshift such that the fliM and fliN genes were placed in frame, coding for an essentially complete FliM-FliN protein fusion. Minicell analysis demonstrated that, as expected, the parental mutant synthesized an essentially full-length FliM protein but no detectable FliN. The pseudorevertant, in contrast, synthesized a protein with the predicted size for the FliM-FliN fusion protein and no detectable FliM or FliN. Immunoblotting of minicells with antibodies against FliM and FliN confirmed the identities of these various proteins. Immunoblotting of book-basal-body complexes from the wild-type strain gave a strong signal for the three switch proteins FliG, FliM, and FliN. Complexes from the FliM-FliN fusion mutant gave a strong signal for FliG but no signal for either FIiM or FliN; a moderately strong signal for the FliM-FliN fusion protein was seen with the anti-FliM antibody, and a weaker signal was seen with the anti-FliN antibody. The cytoplasmic C ring of the structure, which is seen consistently in electron microscopy of wild-type complexes and which is known to contain the FliM and FliN proteins, was much more labile in the FliM-FliN fusion mutant, giving a fragmented and variable appearance or being completely absent. Complementation data indicated that wild-type FliM had a mild dominant negative effect over the fusion protein, that wild-type FliN and the fusion protein work much better than the fusion protein alone, and that wild-type FliM and FliN together have no major positive or negative effect on the function of the fusion protein. We interpret these data to mean that the FliM-FliN fusion protein incorporates into structure but less stably than do the FliM and FliN proteins separately, that wild-type FliM tends to displace the fusion protein, and that wild-type FliN can supplement the FliN domain of the fusion protein without displacing the FliM domain. The data support, but do not prove, a model in which FliM and FliN in the wild-type switch complex are stationary with respect to each other.     

Three-dimensional image reconstruction of straight flagella from a mutant Salmonella typhimurium.

The structure of the straight flagella from a mutant Salmonella typhimurium was studied by electron microscopy using digital image processing, including three-dimensional reconstruction, to an effective resolution of about 14 Å.Three-dimensional studies suggest that there are two sets of intersubunit bonds, i.e. intraprotofilament bonds along the (n = 11, l = 1) helix at a radius of about 55 Å and interprotofilament bonds along the (n = ?5, l = 7) helix at radii of about 10 to 15 Å and 50 Å, and along the (n = 6, l = 8) helix at a radius of about 45 Å and along the (n = 1, l = 15) helix at a radius of about 20 Å. There are four high density regions in a morphological subunit. These regions are situated at radii of about 15 Å, 40 Å, 70 Å and 80 Å. Variation was seen in the position of the high density regions at radii of about 15 Å and 40 Å among the ten models that were reconstituted individually. The regions at radii of 40 Å and 70 Å are the highest in density. The radial distance between these two regions is consistent with the 32 Å feature of a cylindrically averaged Patterson function calculated using equatorial data from X-ray diffraction pattern (Champness, 1968,1971).At the outer radii of the flagellum the shape of the morphological subunit roughly corresponded to that of the “chevron” described by O'Brien &; Bennett (1972), but there was no corresponding structure at the inner radii; the appearance of chevrons in that region might arise from the superposition of the two sides of the helical lattice.The biological significance of the “beaded” submolecular structure of flagellin and the presence of two sets of intersubunit bonds at the different radii is discussed with reference to the waveform and polymorphic behaviour of flagellar filaments.